Brassica plants yielding oils with a low alpha linolenic acid content

ABSTRACT

Brassica  plants producing oils with a low alpha-linolenic acid content and methods for producing such plants are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Patent Application No. PCT/US2011/037864, filed May 25, 2011, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 61/348,121, filed May 25, 2010.

TECHNICAL FIELD

This invention relates to Brassica plants, and more particularly, Brassica plants having a modified allele at a fatty acid desaturase 3D locus and/or a fatty acid desaturase 3E locus and yielding an oil with a low alpha linolenic acid content in combination with a typical, mid, or high oleic acid content.

BACKGROUND

Canola oil contains a relatively high (8%-10%) alpha-linolenic acid (ALA) content. This trienoic fatty acid is unstable and easily oxidized during cooking, which in turn creates off-flavors of the oil. It also develops off odors and rancid flavors during storage (Hawrysh, 1990, Stability of canola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed. Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology, Van Nostrand Reinhold, N.Y.). Reducing the ALA content level by hydrogenation increases oxidative stability of the oil. However, hydrogenation results in the production of trans fatty acids, which increases the risk for coronary heart disease when consumed. Although an oil's oxidative stability is not determined solely by fatty acid profile, a decrease in the ALA content of canola oils generally improves the stability of the oils.

SUMMARY

This document is based on the discovery of a modified fad3D allele and a modified fad3E allele, and use of such alleles in Brassica plants to control ALA content. As described herein, Brassica plants containing such a modified fad3D allele and modified fad3E allele can produce oils with a low ALA content (i.e. 1.5% or less ALA). Such Brassica plants also can include other modified fatty acid desaturase alleles (e.g., fad2 or fad3), fatty acyl-acyl carrier protein thioesterase A2 (fatA2), and/or fatty acyl-acyl carrier protein thioesterase B (fatB) alleles to tailor the oleic acid and total saturated fatty acid content to the desired end use of the oil. Brassica plants described herein are particularly useful for producing canola oils for certain food applications as the plants are not genetically modified.

In one aspect, this document features a Brassica plant (e.g., Brassica napus, Brassica juncea, or Brassica rapa plant), progeny, and seeds of the plant that include a modified allele at a fatty acid desaturase 3D (fad3D) locus and/or a fatty acid desaturase 3E (fad3E) locus, wherein the modified allele results in the production of a FAD3D and/or FAD3E polypeptide having reduced desaturase activity relative to a corresponding wild-type polypeptide. The fad3E modified allele can include a nucleic acid encoding a truncated FAD3E polypeptide, a nucleic acid encoding a FAD3E polypeptide having a non-conservative substitution of a residue affecting substrate specificity, or a nucleic acid encoding a FAD3E polypeptide having a non-conservative substitution of a residue affecting catalytic activity. In some embodiments, the fad3E modified allele includes a mutation in a splice donor site. A modified fad3E allele can include a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. The fad3D modified allele can include a nucleic acid encoding a truncated FAD3D polypeptide, a nucleic acid having a deletion of an exon or a portion thereof (e.g., a deletion within exon 1 of the nucleic acid). In some embodiments, the fad3D modified allele includes a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:32. A plant can include fad3E and fad3D modified alleles. The fad3E and fad3D modified alleles can be mutant alleles. A plant can be an F₁ hybrid.

Any of the plants described herein further can include a modified allele at a fad3A locus and/or a modified allele at a fad3B locus. The fad3A and/or fad3B modified alleles can be mutant alleles. For example, a fad3A modified allele can be selected from the group consisting of a) a nucleic acid encoding a FAD3A polypeptide having a cysteine substituted for arginine at position 275 and b) a nucleic acid encoding a truncated FAD3A polypeptide. A fad3B modified allele can be selected from the group consisting of a) a nucleic acid having a mutation in an exon-intron splice site recognition sequence and b) a nucleic acid encoding a truncated FAD3B polypeptide. Such plants can produce seeds yielding an oil having an ALA content of 0.6 to 1.5%.

Plants described herein can produce seeds yielding an oil having a stearic acid content of 2.5 to 6%.

Any of the plants described herein further can include a modified allele at a delta-12 fatty acid desaturase (fad2) locus. The fad2 modified allele can include a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26). The fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a glutamic acid substituted for glycine in the DRDYGILNKV motif (SEQ ID NO:28) or a histidine substituted for leucine in a KYLNNP motif (SEQ ID NO:27).

Any of the plants described herein further can include a modified allele at two different fad2 loci. One fad2 modified allele can include a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26). One fad2 modified allele can include a nucleic acid encoding a FAD2 polypeptide having a glutamic acid substituted for glycine in the DRDYGILNKV motif (SEQ ID NO:28) or a histidine substituted for leucine in a KYLNNP motif (SEQ ID NO:27).

Any of the plants described herein further can include a modified allele at a fatty acyl-acyl-ACP thioesterase A2 (fatA2) locus and/or a fatty acyl-acyl-ACP thioesterase B (fatB) locus. The fatA2 and/or fatB modified alleles can be mutant alleles. A fatA2 modified allele results in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide. The fatA2 modified allele can include a nucleic acid encoding a FATA2 polypeptide having a mutation in a region (SEQ ID NO:11) corresponding to amino acids 242 to 277 of the FATA2 polypeptide. The FATA2 polypeptide can include a substitution of a leucine residue for proline at position 255. A fatB modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide. A plant can include modified alleles at four different fatB loci. At least one of the fatB modified alleles can include a nucleic acid encoding a truncated FATB polypeptide. For example, a truncated FATB polypeptide can include a nucleotide sequence selected from the group consisting of: SEQ ID NO:22 SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

In another aspect, this document features a method of producing an oil. The method includes crushing seeds produced from at least one Brassica plant described herein; and extracting oil from the crushed seeds, wherein the oil has, after refining, bleaching, and deodorizing, an ALA content of 0.6 to 1.5%. The oil further can have a stearic acid content of 2.5 to 6.0%.

This document also features a method for making a Brassica progeny plant. The method includes crossing one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or a fad3D locus and one or more second Brassica parent plants comprising a modified allele at a different fad3 locus, wherein each modified allele results in the production of a FAD3 polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; and selecting, for one to five generations, for progeny plants having a modified allele at the fad3E locus and/or fad3D locus, and the modified allele at the different fad3 locus, thereby obtaining the Brassica plant.

In another aspect, this document features a method for making a Brassica plant. The method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or modified allele at a fad3D locus, wherein the fad3E or fad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising a modified allele at a fad2 locus, the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glycine in a His-Glu-Cys-Gly-His motif (SEQ ID NO:26); crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for progeny plants having the modified allele at the fad3E locus and/or fad3D locus, and the modified allele at the fad2 locus thereby obtaining the Brassica plant. The first Brassica parent plant can include a modified allele at three different fad3 loci (e.g., fad3D, fad3A and fad3B).

In another aspect, this document features a method for making a Brassica plant. The method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus and/or fad3D locus, wherein the fad3E or said fad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising a modified allele at a fatA2 locus, the fatA2 modified allele comprising a nucleic acid encoding a FATA2 polypeptide having a mutation in a region (SEQ ID NO:11) corresponding to amino acids 242 to 277 of the FADA2 polypeptide; crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for progeny plants having the modified allele at the fad3E locus and/or the fad3D locus, and the modified allele at the fatA2 locus thereby obtaining the Brassica plant. The first Brassica parent plant further can include a modified allele at a fad2 locus, a modified allele at a fad3A locus, and a modified allele at a fad3B locus, wherein the fad2 modified allele comprising a nucleic acid encoding a FAD2 polypeptide having a lysine substituted for glutamic acid in a His-Glu-Cys-Gly-His motif (SEQ ID N:26), the fad3A modified allele comprising a nucleic acid encoding a FAD3A polypeptide having a cysteine substituted for arginine at position 275, and the fad3B modified allele comprising a fad3B nucleic acid sequence having a mutation in an exon-intron splice site recognition sequence.

This document also features a method for making a Brassica plant. The method includes obtaining one or more first Brassica parent plants comprising a modified allele at a fad3E locus or a fad3D locus, wherein the fad3E or fad3D modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; obtaining one or more second Brassica parent plants comprising at least one modified allele at a fatB locus, wherein the fatB modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide; crossing the one or more first Brassica parent plants and the one or more second Brassica parent plants; and selecting, for one to five generations, for progeny plants having the modified allele at the fad3E locus and/or fad3D locus, and the at least one modified fatB allele at the fatB locus, thereby obtaining the Brassica plant. The one or more second Brassica plants can include modified alleles at four different fatB loci. At least one of the fatB modified alleles can include a nucleic acid encoding a truncated FATB polypeptide.

In another aspect, this document features seeds of a Brassica plant comprising a modified allele at a fad3E locus and/or a modified allele at a fad3D locus. The fad3E modified allele can include a nucleic acid having a mutation in a splice donor site. The fad3D modified allele can include a nucleic acid having a deletion of a portion of exon 1. The seeds can yield an oil having an ALA content of 0.6% to 1.5%. The seeds can be F₂ seeds. The Brassica plant further can include modified alleles at fad3A and/or fad3B loci. The Brassica plant further can include a modified allele at a fad2 locus. The Brassica plant further can include a modified allele at a fatB locus. The Brassica plant further can include a modified allele at a fatA2 locus. The Brassica plant further can include modified alleles at fad3A, fad3B, fad2, fatB, and fatA2 loci.

In yet another aspect, this document features a plant cell of a plant described herein, wherein the plant cell includes one or more of the modified alleles.

This document also features an isolated nucleic acid that includes a nucleic acid sequence selected from the group consisting of i) the nucleic acid sequence set forth in SEQ ID NO:1; ii) the nucleic acid sequence set forth in SEQ ID NO:32; iii) the complement of the nucleic acid sequence set forth in i) or ii); and iv) a nucleic acid fragment of i), ii), or iii) that is at least 50 nucleotides in length and distinguishes a mutant fad3D or fad3E allele from a wild-type fad3D or fad3E allele.

In another aspect, this document features a method of making a plant line. The method includes providing a population of plants; identifying one or more plants in the population containing a modified allele at a fad3E locus and/or a fad3D locus, wherein the modified allele results in the production of a FAD3E or FAD3D polypeptide having reduced desaturase activity relative to a corresponding wild-type FAD3 polypeptide; crossing one or more of the identified plants with itself or a different plant to produce seed; crossing at least one progeny plant grown from the seed with itself or a different plant; and repeating the crossing steps for an additional 0-5 generations to make the plant line, wherein the modified allele at the fad3E locus and/or the fad3D locus is present in the plant line.

This document also features Brassica napus seed designated 1904 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11273, as well as progeny of the seed designated 1904 and represented by ATCC Accession No. PTA-11273.

This document also features Brassica napus seed designated 2558 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11274, as well as progeny of the seed designated 2558 and represented by ATCC Accession No. PTA-11274.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All numbers expressing quantities of ingredients, properties such as molecular weight, percentages, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth are approximations that may depend upon the desired properties sought.

Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an alignment of the BnFad3E sequences from 1904, IMC201, and BrFad3E (SEQ ID NOs:1, 2, and 3, respectively). The BnFad3E-2 SNP that correlates with the low ALA (C18:3) content in the 1904 mutant line is highlighted with a black box at position 1851 of this alignment. At the position 1756 in SEQ ID NO:1 (1904 BnFad3E-2.seq), a single nucleotide mutation (G to A) is shown, which is located in a splice donor site (see FIG. 2). The start codon (ATG) is underlined at position 94 of this sequence alignment and the stop codon in BrFad3E (TAA) is at position 3828.

FIG. 2 is an alignment of the nucleotide sequence of the exon 3, intron 3 border of the BnFad3A, BnFad3B, BnFad3E genes from IMC201, IMC02, Westar, 1904, 2558, and 95CB504, and the BrFad3E gene from Brassica rapa (world wide web at brassica-rapa.org) showing the single nucleotide mutation (G to A) in BnFad3E-2 from the 1904 line. See SEQ ID NOs:4-8. This mutation (G to A) is located in the last nucleotide of exon 3 of BnFad3E. Intron 3 of the BnFad3E starts from the sequence GT (see SEQ ID NO:8).

FIG. 3 is an alignment of the amino acid sequences of FAD3E polypeptides from B. napus and B. rapa, and FAD3 from Arabidopsis thaliana. See SEQ ID NOs:29, 30, and 31.

FIG. 4 is an alignment of the BnFad3D sequences from 1904 (SEQ ID NO:32) and IMC201 and 95CB504 (SEQ ID NO:33) showing a DNA deletion in the 1904 BnFad3D starting at position 575 in this alignment, which includes a portion of exon 1 and intron 1. The start codon (ATG) is at position 441.

FIG. 5 is the amino acid sequence of the FAD3D polypeptide (SEQ ID NO:34). In line 1904, the FAD3D polypeptide is truncated after amino acid 64.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In general, this document provides Brassica plants, including B. napus, B. juncea, and B. rapa species of Brassica, that yield seeds producing oils having a low ALA content (i.e., 1.6% or less). Canola oil produced from seeds having a low ALA content tends to exhibit increased stability (e.g., oxidative stability and/or flavor stability) and a useful nutritional profile, and can be used for many food applications including as a frying oil.

In some embodiments, plants described herein yield seeds producing oils having a low ALA content in combination with low total saturated fatty acids (i.e., 6% or less) or very low total saturated fatty acids (i.e., having 3.6% or less). As used herein, total saturated fatty acid content refers to the total of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0). For example, Brassica plants described herein can produce oils having a low ALA content and a total saturated fatty acid content of 2.5 to 6.0%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to 3.5%, 3.4 to 3.7%, 3.6 to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%. Oils having a low ALA content and a low or very low total saturated fatty acid content have improved oxidative stability and nutritional quality and can help consumers reduce their intake of saturated fatty acids.

In some embodiments, Brassica plants yield seed oils having a low ALA content in combination with a typical (60%-70%), mid (70.1%-80%), or high (>80%) oleic acid content. In some embodiments, the total saturated fatty acid content of such seed oils can be less than 6%. As such, Brassica plants can produce seed oils having a fatty acid content tailored to the desired end use of the oil (e.g., frying or food applications). For example, Brassica plants can be produced that yield seeds having a low ALA content (e.g., 1.5% or less), an oleic acid content of 60 to 70%, and a linoleic acid content of 17 to 24%. Canola oils having such a fatty acid profile are particularly useful for frying applications due to the polyunsaturated fatty acid content, which is low enough to have improved oxidative stability for frying yet high enough to impart the desired fried flavor to the food being fried, and are an improvement over commodity type canola oils. The fatty acid content of commodity type canola oils may be on the order of is 6 to 8% total saturated fatty acids, 55 to 65% oleic acid, 20 to 30% linoleic acid, and 7 to 10% α-linolenic acid. See, e.g., Bailey's Industrial Oil and Fat Products, Section 2.2, “Canola Oil” on pages 61-121 of Volume 2 (6th Edition, 2005).

In some embodiments, Brassica plants can be produced that yield seeds having a low ALA content, mid-oleic acid content (e.g., 70.1 to 80% oleic acid) and a low total saturated fatty acid content (e.g., <6.0%). Canola oils having such a fatty acid profile have an oxidative stability that is higher than oils with higher ALA and lower oleic acid contents or commodity type canola oils, and are useful for coating applications (e.g., spray-coatings), formulating food products, or other applications where shelf-life stability is desired. In addition, Brassica plants can be produced that yield seeds having a low ALA content, high oleic acid content (e.g., 80.1 to 90% oleic acid) and a low total saturated fatty acid content (<6.0%). Canola oils having a low ALA, high oleic acid, and low total saturated fatty acid content are particularly useful for food applications requiring high oxidative stability and a reduced saturated fatty acid content.

Brassica Plants

Brassica plants described herein can have reduced levels of ALA (e.g., 8.0% or less) in the seed oil as a result of reduced activity of fatty acid desaturase (FAD) 3E (also known as delta-15 desaturase). Brassica plants described herein also can have reduced levels of ALA (e.g., 3.0% or less, 2.8% or less, 2.6% or less) in the seed oil as a result of reduced activity of FAD3D. FAD3 proteins are involved in the enzymatic conversion of linoleic acid to α-linolenic acid. Sequences of higher plant Fad3 genes are disclosed in Yadav et al., Plant Physiol., 103:467-476 (1993), WO 93/11245, and Arondel et al., Science, 258:1353-1355 (1992). It is understood that throughout the disclosure, reference to “plant” or “plants” includes progeny, i.e., descendants of a particular plant or plant line, as well as cells or tissues from the plant. Progeny of an instant plant include seeds formed on F₁, F₂, F₃, F₄ and subsequent generation plants, or seeds formed on BC₁, BC₂, BC₃, and subsequent generation plants. Seeds produced by a plant can be grown and then selfed (or outcrossed and selfed, or doubled through dihaploid) to obtain seeds homozygous for a modified allele. The term “allele” or “alleles” refers to one or more alternative forms of a gene at a particular locus. As used herein, a “line” is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques. As used herein, the term “variety” refers to a line which is used for commercial production, and includes hybrid varieties and open-pollinated varieties.

Reduced activity, including absence of detectable desaturase activity, of FAD3E and/or FAD3D can be achieved by modifying an endogenous fad3E or fad3D allele. An endogenous fad3E or fad3D allele can be modified by, for example, mutagenesis or by using homologous recombination to replace an endogenous plant gene with a variant containing one or more mutations (e.g., produced using site-directed mutagenesis). See, e.g., Townsend et al., Nature 459:442-445 (2009); Tovkach et al., Plant J., 57:747-757 (2009); and Lloyd et al., Proc. Natl. Acad. Sci. USA, 102:2232-2237 (2005). Similarly, for other genes discussed herein, the endogenous allele can be modified by mutagenesis or by using homologous recombination to replace an endogenous gene with a variant. Modified alleles obtained through mutagenesis are encompassed by the term “mutant alleles” as that term is used herein.

Reduced desaturase activity, including absence of detectable activity, can be inferred from the decreased level of linolenic acid (product) and in some cases, increased level of linoleic acid (the substrate) in the plant compared with a corresponding control plant. Reduced activity also can be assessed by in vitro translation of the desaturase and assaying for desaturase activity. See, for example, Goren and Fox, Protein Expr Purif. 62(2): 171-178 (2008).

Genetic mutations can be introduced within a population of seeds or regenerable plant tissue using one or more mutagenic agents. Suitable mutagenic agents include, for example, ethyl methane sulfonate (EMS), methyl N-nitrosoguanidine (MNNG), ethidium bromide, diepoxybutane, ionizing radiation, x-rays, UV rays and other mutagens known in the art. In some embodiments, a combination of mutagens, such as EMS and MNNG, can be used to induce mutagenesis. The treated population, or a subsequent generation of that population, can be screened for reduced desaturase activity that results from the mutation, e.g., by determining the fatty acid profile of the population and comparing it to that of a corresponding non-mutagenized population. Mutations can be in any portion of a gene, including coding sequence, exon sequence, intron sequence, and regulatory elements, that render the resulting gene product non-functional or with reduced activity. Suitable types of mutations include, for example, insertions or deletions of nucleotides, and transitions or transversions in the wild-type coding sequence. Such mutations can lead to deletion or insertion of amino acids, and conservative or non-conservative amino acid substitutions in the corresponding gene product. In some embodiments, the mutation is a deletion of an exon or a portion thereof, resulting in the production of a truncated polypeptide from either lack of or incorrect RNA splicing. In some embodiments, the mutation is a nonsense mutation, which results in the introduction of a stop codon (TGA, TAA, or TAG) and production of a truncated polypeptide. The gene product of an allele having a stop codon mutation typically lacks detectable desaturase activity. In some embodiments, the mutation is a splice site mutation which alters or abolishes the correct splicing of the pre-mRNA sequence, resulting in a protein of different amino acid sequence than the wild type. For example, one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons. Alternatively, the reading frame may be altered by incorrect splicing, one or more introns may be retained, alternate splice donors or acceptors may be generated, or splicing may be initiated at an alternate position, or alternative polyadenylation signals may be generated. In some embodiments, more than one mutation or more than one type of mutation is introduced. PCR can be used to amplify modified alleles in genomic DNA from the plant or plant tissue, and the resulting amplification product can be isolated and sequenced to characterize the polypeptide encoded by the modified allele. In some embodiments, RT-PCR can be used to detect particular RNA transcripts.

Insertions, deletions, or substitutions of amino acids in a protein sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions, deletions, or substitutions also can disrupt binding, alter substrate specificity, or disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue.

Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in reduced activity may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.

In some embodiments, a plant described herein contains a modified allele at a fad3E locus. For example, a fad3E locus can include a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO:1. The nucleotide sequence set forth in SEQ ID NO:1 is a representative nucleotide sequence of the fad3E gene from B. napus line 1904, which contains a single nucleotide mutation (G to A) in a splice donor site. As used herein, the term “sequence identity” refers to the degree of similarity between any given nucleic acid sequence and a target nucleic acid sequence. The degree of similarity is represented as percent sequence identity. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity also can be determined for any amino acid sequence. Percent sequence identity can be determined using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at “fr” dot “com” slash “blast”) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at “ncbi” dot “nlm” dot “nih” dot “gov”). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ.

Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and all other options are left at their default setting. The following command will generate an output file containing a comparison between two sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1-r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.

Once aligned, a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide is presented in both the target and identified sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.

The percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (i) a 500-base nucleic acid target sequence is compared to a subject nucleic acid sequence, (ii) the Bl2seq program presents 200 bases from the target sequence aligned with a region of the subject sequence where the first and last bases of that 200-base region are matches, and (iii) the number of matches over those 200 aligned bases is 180, then the 500-base nucleic acid target sequence contains a length of 200 and a sequence identity over that length of 90% (i.e., 180, 200×100=90).

It will be appreciated that different regions within a single nucleic acid target sequence that aligns with an identified sequence can each have their own percent identity. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.

In some embodiments, a plant described herein contains a modified allele at a fad3D locus. For example, a fad3D locus can include a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO:32. The nucleotide sequence set forth in SEQ ID NO:32 is a representative nucleotide sequence of the fad3D gene from B. napus line 1904, which contains a deletion of 164 nucleotides from exon 1. In B. napus line IMC201 and 95CB504, exon 1 starts at position 441 and ends at position 739. See, e.g., FIG. 4.

In some embodiments, a Brassica plant contains a modified fad3E allele and a modified fad3D allele. A modified fad3E and a modified fad3D allele may be combined in a plant by making a genetic cross between modified lines. For example, a plant having a modified allele at a fad3E locus can be crossed or mated with a second plant having a modified allele at a fad3D locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both modified alleles. In some embodiments, progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified fad3E and fad3D alleles. In some embodiments, a line having both fad3E and fad3D modified alleles is used to introgress an individual modified allele into a different line or to introgress both modified alleles into a different line.

In some embodiments, a Brassica plant contains a modified fad3E allele or a modified fad3D allele, and optionally one or more modified alleles at fad3 (e.g. fad3A and/or fad3B), fatA2, fatB, and fad2 loci. In some embodiments, a Brassica plant contains a modified fad3E allele and a modified fad3D allele, and optionally one or more modified alleles at fad3 (e.g., fad3A and/or fad3B), fatA2, fatB, and fad2 loci. For example, a Brassica plant can contain a modified fad3E allele, a modified fad3D allele, and one or more other modified fad3 alleles. For example, in addition to a modified fad3E and fad3D allele, Brassica plants can contain the mutation from the APOLLO or STELLAR B. napus variety that confers low linolenic acid. The STELLAR and APOLLO varieties were developed at the University of Manitoba (Manitoba, Canada). In some embodiments, the disclosed plants contain the fad3A and/or fad3B mutation from IMC02 that confer a low linolenic acid phenotype. IMC02 contains a mutation in both the fad3A and fad3B genes. The fad3A gene contains a C to T mutation at position 2565, numbered from the ATG in genomic DNA, resulting in the substitution of a cysteine for arginine at position 275 of the encoded FAD3A polypeptide. The fad3B gene contains a G to A mutation at position 3053 from ATG in genomic DNA, located in the exon-intron splice site recognition sequence. IMC02 was obtained from a cross of IMC01 x Westar. See Example 3 of U.S. Pat. No. 5,750,827. IMC01 was deposited with the American Type Culture Collection (ATCC) under Accession No. 40579. IMC02 was deposited with the ATCC under Accession No. PTA-6221. Other examples of fad3 mutations include nonsense mutations in fad3A and fad3B sequences. See, Example 4. For example, the mutant fad3A sequence set forth in SEQ ID NO:9 contains a mutation at position 102, resulting in a codon change from TGG to TGA and production of a truncated FAD3A polypeptide. The mutant fad3B sequence set forth in SEQ ID NO:10 contains a mutation at position 206, resulting in a codon change from TGG to TAG and production of a truncated FAD3B polypeptide.

Two or more different modified fad3 alleles may be combined in a plant by making a genetic cross between modified lines. For example, a plant having a modified allele at a fad3E locus and/or a fad3D locus can be crossed or mated with a second plant having a modified allele at a fad3A or fad3B locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both modified alleles. In some embodiments, progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified alleles at two different fad3 loci.

Brassica plants having a modified allele at a fad3E locus or fad3D locus also can include modified alleles controlling fatty acyl-ACP thioesterase A2 (fatA2) and/or fatty acyl-ACP thioesterase B (fatB) to tailor the total saturated fatty acid content to the end use of the oil. Fatty acyl-ACP thioesterases hydrolyze acyl-ACPs in the chloroplast to release the newly synthesized fatty acid from ACP, effectively removing it from further chain elongation in the plastid. The free fatty acid can then leave the plastid, become bound to CoenzymeA (CoA) and enter the Kennedy pathway in the endoplasmic reticulum (ER) for triacylglycerol (TAG) biosynthesis. Members of the FATA family prefer oleoyl (C18:1) ACP substrates with minor activity towards 18:0 and 16:0-ACPs, while members of the FATB family hydrolyze primarily saturated acyl-ACPs between 8 and 18 carbons in length. See Jones et al., Plant Cell 7:359-371 (1995); Ginalski and Rhchlewski, Nucleic Acids Res 31:3291-3292 (2003); and Voelker T in Genetic Engineering (Setlow, J K, ed) Vol 18, 111-133, Plenum Publishing Corp., New York (2003).

Reduced activity of FATA2 and/or FATB, including absence of detectable activity, can be inferred from the decreased level of saturated fatty acids in the seed oil compared with seed oil from a corresponding control plant. Reduced activity also can be assessed in plant extracts using assays for fatty acyl-ACP hydrolysis. See, for example, Bonaventure et al., Plant Cell 15:1020-1033 (2003); and Eccleston and Ohlrogge, Plant Cell 10:613-622 (1998).

In some embodiments, in addition to a modified allele at a fad3E locus and/or a fad3D locus, and optionally one or more other modified fad3 loci, a Brassica plant contains a modified allele at a fatA2 locus, wherein the modified allele results in the production of a FATA2 polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATA2 polypeptide. For example, the modified fatA2 allele can include a nucleic acid that encodes a FATA2 polypeptide having a non-conservative substitution within a helix/4-stranded sheet (4HBT) domain (also referred to as a hot-dog domain) or non-conservative substitution of a residue affecting catalytic activity or substrate specificity. For example, a Brassica plant can contain a modified allele that includes a nucleic acid encoding a FATA2b polypeptide having a substitution in a region (SEQ ID NO:11) of the polypeptide corresponding to residues 242 to 277 of the FATA2 polypeptide (as numbered based on the alignment to the Arabidopsis thaliana FATA2 polypeptide set forth in GenBank Accession No. NP_(—)193041.1, protein (SEQ ID NO:12); GenBank Accession No. NM_(—)117374, mRNA). This region of FATA2 is highly conserved in Arabidopsis and Brassica. In addition, many residues in this region are conserved between FATA and FATB, including the aspartic acid at position 259, asparagine at position 263, histidine at position 265, valine at position 266, asparagine at position 268, and tyrosine at position 271 (as numbered based on the alignment to SEQ ID NO:12). The asparagine at position 263 and histidine at position 265 are part of the catalytic triad, and the arginine at position 256 is involved in determining substrate specificity. See also Mayer and Shanklin, BMC Plant Biology 7:1-11 (2007). SEQ ID NO:13 sets forth the predicted amino acid sequence of the Brassica FATA2b polypeptide encoded by exons 2-6, and corresponding to residues 121 to 343 of the A. thaliana sequence set forth in SEQ ID NO:12. For example, the FATA2 polypeptide can have a substitution of a leucine residue for proline at the position corresponding to position 255 of the Arabidopsis FATA2 polypeptide (i.e., position 14 of SEQ ID NO:11 or position 135 of SEQ ID NO: 13). The proline in the B. napus sequence corresponding to position 255 in Arabidopsis is conserved among B. napus, B. rapa, B. juncea, Zea mays, Sorghum bicolor, Oryza sativa Indica (rice), Triticum aestivum, Glycine max, Jatropha (tree species), Carthamus tinctorius, Cuphea hookeriana, Iris tectorum, Perilla frutescens, Helianthus annuus, Garcinia mangostana, Picea sitchensis, Physcomitrella patens subsp. Patens, Elaeis guineensis, mitis vinifera, Elaeis oleifera, Camellia oleifera, Arachis hypogaea, Capsicum annuum, Cuphea hookeriana, Populus trichocarpa, and Diploknema butyracea. The mutation at position 255 is associated with a low total saturated fatty acid phenotype, low stearic acid phenotype, low arachidic acid phenotype, and an increased eicosenoic acid phenotype. The stearic acid content phenotype is negatively correlated with the eicosenoic acid phenotype. See, U.S. Provisional Application Nos. 61/287,985 and 61/295,049.

In some embodiments, the modified allele at a fatA2 locus includes a nucleotide sequence having at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) sequence identity to the nucleotide sequence set forth in SEQ ID NO:14 or SEQ ID NO:15. The nucleotide sequences set forth in SEQ ID NOs:14 and 15 are representative nucleotide sequences from the mutant fatA2b gene from B. napus line 15.24. See, U.S. Provisional Application Nos. 61/287,985 and 61/295,049.

In some embodiments, a Brassica plant contains a modified allele at a fatB locus, wherein the modified allele results in the production of a FATB polypeptide having reduced thioesterase activity relative to a corresponding wild-type FATB polypeptide. In some embodiments, a Brassica plant contains modified alleles at two or more different fatB loci. In some embodiments, a Brassica plant contains modified alleles at three different fatB loci or contains modified alleles at four different fatB loci. Brassica napus contains 6 different FATB isoforms (i.e., different forms of the FATB polypeptide at different loci), which are called isoforms 1-6 herein. SEQ ID NOs:16-21 set forth the nucleotide sequences encoding FATB isoforms 1-6, respectively, of Brassica napus. The nucleotide sequences set forth in SEQ ID NOs:16-21 have 82% to 95% sequence identity as measured by the ClustalW algorithm (version 1.83, default parameters). See Chenna et al., Nucleic Acids Res., 31(13):3497-500 (2003).

For example, in addition to a modified allele at a fad3E locus and a fad3D locus, a Brassica plant can have a mutation in a nucleotide sequence encoding FATB isoform 1, isoform 2, isoform 3, isoform 4, isoform 5, or isoform 6. In some embodiments, a plant can have a mutation in a nucleotide sequence encoding FAD3E and can have mutation in a nucleotide sequence encoding 2 or more FATB isoforms, e.g., FATB isoforms 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 2 and 3; 2 and 4; 2 and 5; 2 and 6; 3 and 4; 3 and 5; 3 and 6; 4 and 5; 4 and 6; 5 and 6; 1, 2, and 3; 1, 2, and 4; 1, 2, and 5; 1, 2, and 6; 1, 3, and 4; 1, 3, and 5; 1, 3, and 6; 1, 4, and 5; 1, 4, and 6; 1, 5, and 6; 2, 3, and 4; 2, 3, and 5; 2, 3, and 6; 2, 4, and 5; 2, 4, and 6; 1, 5, and 6; 3, 4, and 5; 3, 4, and 6; 3, 5, and 6; 4, 5, and 6; 1, 2, 3, and 4; 1, 2, 3, and 5; 1, 2, 3, and 6; 1, 2, 4, and 5; 1, 2, 4, and 6; 1, 2, 5, and 6; 1, 3, 4 and 5; 1, 3, 4, and 6; 1, 3, 5, and 6; 1, 4, 5, and 6; 2, 3, 4, and 5; 2, 3, 4 and 6; 2, 3, 5, and 6; 2, 4, 5, and 6; or 3, 4, 5, and 6. In some embodiments, a Brassica plant can have a mutation in a nucleotide sequence encoding a FAD3E polypeptide and can have a mutation in nucleotide sequences encoding FATB isoforms 1, 2, and 3; 1, 2, and 4; 1, 3, and 4; 2, 3, and 4; or 1, 2, 3, and 4. In some embodiments, a mutation in a FATB isoform results in deletion of a 4HBT domain or a portion thereof of a FATB polypeptide. FATB polypeptides typically contain a tandem repeat of the 4HBT domain, where the N-terminal 4HBT domain contains residues affecting substrate specificity (e.g., two conserved methionines, a conserved lysine, a conserved valine, and a conserved serine) and the C-terminal 4HBT domain contains residues affecting catalytic activity (e.g., a catalytic triad of a conserved asparagine, a conserved histidine, and a conserved cysteine) and substrate specificity (e.g., a conserved tryptophan). See Mayer and Shanklin, J. Biol. Chem. 280:3621-3627 (2005). In some embodiments, the mutation in a nucleotide sequence encoding FATB results in a non-conservative substitution of a residue in a 4HBT domain or a residue affecting substrate specificity. In some embodiments, the mutation in a nucleotide sequence encoding FATB is a splice site mutation. In some embodiment, the mutation in a nucleotide sequence encoding FATB is a nonsense mutation in which a premature stop codon (TGA, TAA, or TAG) is introduced, resulting in the production of a truncated polypeptide.

SEQ ID NOs:22-25 set forth the nucleotide sequences encoding fatB isoforms 1-4, respectively, and containing exemplary nonsense mutations that result in truncated FATB polypeptides. SEQ ID NO:22 is the nucleotide sequence of isoform 1 having a mutation at position 154, which changes the codon from CAG to TAG. SEQ ID NO:23 is the nucleotide sequence of isoform 2 having a mutation at position 695, which changes the codon from CAG to TAG. SEQ ID NO:24 is the nucleotide sequence of isoform 3 having a mutation at position 276, which changes the codon from TGG to TGA. SEQ ID NO:25 is the nucleotide sequence of isoform 4 having a mutation at position 336, which changes the codon from TGG to TGA. See also U.S. Provisional Application Nos. 61/287,985 and 61/295,049.

Two or more different modified FATB alleles may be combined in a plant by making a genetic cross between modified lines. For example, a plant having a modified allele at a FATB locus encoding isoform 1 can be crossed or mated with a second plant having a modified allele at a FATB locus encoding isoform 2. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying both modified alleles. In some embodiments, progeny are selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having modified alleles at two different FATB loci. Similarly, a plant having modified alleles at two or more different FATB isoforms can be crossed with a second plant having modified alleles at two or more different FATB alleles, and progeny seeds can be screened to identify those seeds carrying modified alleles at four or more different FATB loci. Again, progeny can be selected for multiple generations to obtain the desired plant.

In some embodiments, a modified allele at a fad3E locus, a fad3D locus, a fatA2 locus and modified alleles at two or more (e.g., three or four) different fatB loci can be combined in a plant. For example, a plant having a modified allele at a fad3E locus and a fad3D locus can be crossed or mated with a second plant having a modified allele at a fatA2 locus. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying modified fad3E, fad3D, and fatA2 alleles. Progeny can be selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having a modified allele at a fad3E locus, a modified allele at a fad3D locus, and a modified allele at a fatA2 locus. Furthermore, progeny identified as having a modified allele at a fad3E locus, a modified allele at a fad3D locus, and a modified allele at a fatA2 locus can be crossed or mated with a second plant having modified alleles at two or more different fatB loci. Seeds produced from the cross are planted and the resulting plants are selfed in order to obtain progeny seeds. These progeny seeds can be screened in order to identify those seeds carrying modified fad3E, fad3D, fatA2, and fatB alleles. Progeny can be selected over multiple generations (e.g., 2 to 5 generations) to obtain plants having a modified allele at a fad3E locus, a modified allele at a fatA2 locus, and two or more different fatB loci. Plants having a modified allele at a fad3E locus, a fad3D locus, a fatA2b locus, and modified alleles at three or four different fatB loci have a low ALA content, high oleic acid content, and a low total saturated fatty acid content.

Brassica plants described herein also can have decreased activity of a delta-12 desaturase, which is involved in the enzymatic conversion of oleic acid to linoleic acid, to confer a mid or high oleic acid content in the seed oil. Brassica plants can exhibit reduced activity of delta-12 desaturase (also known as FAD2) in combination with reduced activity of FAD3E and optionally one or more of FAD3A, FAD3B, FATA2, and FATB. The sequences for the wild-type fad2 genes from B. napus (termed the D form and the F form) are disclosed in WO 98/56239. A reduction in delta-12 desaturase activity, including absence of detectable activity, can be achieved by mutagenesis. Decreased delta-12 desaturase activity can be inferred from the decrease level of linoleic acid (product) and increased level of oleic acid (substrate) in the plant compared with a corresponding control plant. Non-limiting examples of suitable fad2 mutations include the G to A mutation at nucleotide 316 within the fad2-D gene, which results in the substitution of a lysine residue for glutamic acid in a HECGH (SEQ ID NO:26) motif. Such a mutation is found within the line IMC129, which has been deposited with the ATCC under Accession No. 40811. Another suitable fad2 mutation can be the T to A mutation at nucleotide 515 of the fad2-F gene, which results in the substitution of a histidine residue for leucine in a KYLNNP (SEQ ID NO:27) motif (amino acid 172 of the Fad2 F polypeptide). Such a mutation is found within the variety Q508. See U.S. Pat. No. 6,342,658. Another example of a fad2 mutation is the G to A mutation at nucleotide 908 of the fad2-F gene, which results in the substitution of a glutamic acid for glycine in the DRDYGILNKV (SEQ ID NO:28) motif (amino acid 303 of the Fad2 F polypeptide). Such a mutation is found within the line Q4275, which has been deposited with the ATCC under Accession No. 97569. See U.S. Pat. No. 6,342,658. Another example of a suitable fad2 mutation can be the C to T mutation at nucleotide 1001 of the fad2-F gene (as numbered from the ATG), which results in the substitution of an isoleucine for threonine (amino acid 334 of the Fad2 F polypeptide). Such a mutation is found within the high oleic acid line Q7415.

Typically, the presence of one of the fad2-D or fad2-F mutations confers a mid-oleic acid phenotype (e.g., 70-80% oleic acid) to the seed oil, while the presence of both fad2-D and fad2-F mutations confers a higher oleic acid phenotype (e.g., >80% oleic acid). For example, Q4275 contains the fad2-D mutation from IMC129 and a fad2-F mutation at amino acid 303. Q508 contains fad2-D mutation from IMC129 and a fad2-F mutation at amino acid 172. Q7415 contains the fad2-D mutation from IMC129 and a fad2-F mutation at amino acid 334. The presence of both fad2 mutations in Q4275, Q508, and Q7415 confers a high oleic acid phenotype of greater than 80% oleic acid.

Thus, in some embodiments, a Brassica plant contains a modified allele at a fad3E locus and a modified allele at a fad2 locus. For example, a Brassica plant can contain a modified allele at a fad3E locus and a modified allele at a fad2 locus described above. A Brassica plant also can contain a modified allele at a fad3E locus, a modified allele at a fad2 locus, and a modified allele at a fatA2 locus. A Brassica plant can contain a modified allele at a fad3E locus, modified alleles at two or more different fatB loci (three or four different loci), and a fad2 locus described above. A Brassica plant also can contain a modified allele at a fad3E locus, fatA2 locus, modified alleles at two or more different fatB loci (three or four different loci) and a modified allele at a fad2 locus described above. In some embodiments, a Brassica plant contains a modified allele at a fad3E locus, at least one modified allele at a different fad3 locus, a modified allele at a fatA2 locus, a modified allele at one or more different fatB loci (e.g., two or more), and a modified allele at one or more fad2 loci. A Brassica plant also can contain modified alleles at a fad3E locus and a fad3D locus, modified alleles at two or more different fatB loci (three or four different loci), modified alleles at fad2 loci, and modified alleles at fad3A and/or fad3B loci described above. A Brassica plant also contain a modified allele at a fad3E locus, a modified allele at a fad3D locus, a modified allele at a fatA2 locus, modified alleles at two or more different FATB loci (three or four different loci), modified alleles at fad2 loci, and modified alleles at fad3A and fad3B loci described above.

One commercially important Brassica crop is B. napus. Commercial B. napus lines may be classified as either spring lines or winter lines. Winter lines are commonly planted in the autumn and flower in the spring after a period of vernalization over the winter. Spring lines do not require vernalization to flower and are commonly planted and harvested in the same growing season. Winter lines are common in Europe, but most winter lines fare poorly in the colder winters of Canada and the northern United States. As a consequence, most B. napus grown commercially in North America are spring lines. One useful embodiment provides a Brassica plant that is a B. napus plant. Though the B. napus plant may have a winter flowering habit, one preferred implementation has a spring growing habit, i.e., it does not require vernalization to flower.

Production of Hybrid Brassica Varieties

Hybrid Brassica varieties can be produced by preventing self-pollination of female parent plants (i.e., seed parents), permitting pollen from male parent plants to fertilize such female parent plants, and allowing F₁ hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be cytoplasmic male sterility (CMS), nuclear male sterility, molecular male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or be produced by self-incompatibility. Female parent plants containing CMS are particularly useful. CMS can be, for example of the ogu (Ogura), nap, pol, tour, or mur type. See, for example, Pellan-Delourme and Renard, 1987, Proc. 7^(th) Int. Rapeseed Conf., Poznan, Poland, p. 199-203 and Pellan-Delourme and Renard, 1988, Genome 30:234-238, for a description of Ogura type CMS. See, Riungu and McVetty, 2003, Can. J. Plant Sci., 83:261-269 for a description of nap, pol, tour, and mur type CMS.

In embodiments in which the female parent plants are CMS, the male parent plants typically contain a fertility restorer gene to ensure that the F₁ hybrids are fertile. For example, when the female parent contains an Ogura type CMS, a male parent is used that contains a fertility restorer gene that can overcome the Ogura type CMS. Non-limiting examples of such fertility restorer genes include the Kosena type fertility restorer gene (U.S. Pat. No. 5,644,066) and Ogura fertility restorer genes (U.S. Pat. Nos. 6,229,072 and 6,392,127). In other embodiments in which the female parents are CMS, male parents can be used that do not contain a fertility restorer. F₁ hybrids produced from such parents are male sterile. Male sterile hybrid seed can be inter-planted with male fertile seed to provide pollen for seed-set on the resulting male sterile plants.

The methods described herein can be used to form single-cross Brassica F₁ hybrids. In such embodiments, the parent plants can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants. The F₁ seed formed on the female parent plants is selectively harvested by conventional means. One also can grow the two parent plants in bulk and harvest a blend of F₁ hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination. Alternatively, three-way crosses can be carried out wherein a single-cross F₁ hybrid is used as a female parent and is crossed with a different male parent that satisfies the fatty acid parameters for the female parent of the first cross. Here, assuming a bulk planting, the overall oleic acid content of the vegetable oil may be reduced over that of a single-cross hybrid; however, the seed yield will be further enhanced in view of the good agronomic performance of both parents when making the second cross. As another alternative, double-cross hybrids can be created wherein the F₁ progeny of two different single-crosses are themselves crossed. Self-incompatibility can be used to particular advantage to prevent self-pollination of female parents when forming a double-cross hybrid.

Hybrids described herein have good agronomic properties and exhibit hybrid vigor, which results in seed yields that exceed that of either parent used in the formation of the F₁ hybrid. For example, yield can be at least 10% (e.g., 10% to 20%, 10% to 15%, 15% to 20%, or 25% to 35%) above that of either one or both parents. In some embodiments, the yield exceeds that of open-pollinated spring canola varieties such as 46A65 (Pioneer) or Q2 (University of Alberta), when grown under similar growing conditions. For example, yield can be at least 10% (e.g., 10% to 15% or 15% to 20%) above that of an open-pollinated variety.

Hybrids described herein typically produce seeds having very low levels of glucosinolates (<30 μmol/gram of de-fatted meal at a moisture content of 8.5%). In particular, hybrids can produce seeds having <20 μmol of glucosinolates/gram of de-fatted meal. As such, hybrids can incorporate mutations that confer low glucosinolate levels. See, for example, U.S. Pat. No. 5,866,762. Glucosinolate levels can be determined in accordance with known techniques, including high performance liquid chromatography (HPLC), as described in ISO 9167-1:1992(E), for quantification of total, intact glucosinolates, and gas-liquid chromatography for quantification of trimethylsilyl (TMS) derivatives of extracted and purified desulfoglucosinolates. Both the HPLC and TMS methods for determining glucosinolate levels analyze de-fatted or oil-free meal.

Canola Oil

Brassica plants disclosed herein are useful for producing canola oils with low ALA content. For example, oil obtained from seeds of Brassica plants described herein may have an ALA content of 0.5% to 1.6% (e.g., 0.5 to 1.5%, 0.5 to 1.0%, 0.5 to 0.8%, 0.6 to 1.4%, 0.6 to 1.3%, 0.6 to 1.2%, 0.6 to 1.1%, 0.6 to 1.0%, 0.6 to 0.8%, 0.7 to 1.2%, 0.7 to 1.1%, 0.8 to 1.2%, or 0.8 to 1.0%). In some embodiments, Brassica plants described herein produce canola oils with low ALA content (e.g., 0.5 to 1.6%) and low or no total saturated fatty acids. For example, oil obtained from seeds of Brassica plants described herein may have an ALA content of 0.5 to 1.5% and a total saturated fatty acid content of 2.5 to 6%, 3 to 5%, 3 to 4.5%, 3.25 to 3.75%, 3.0 to 3.5%, 3.4 to 3.7%, 3.6 to 5%, 4 to 5.5%, 4 to 5%, or 4.25 to 5.25%. The palmitic acid content of such oils can be 2.4 to 3.5% (e.g., 2.5 to 3% or 2.7 to 3.3%). The stearic acid content of such oils can be 0.7 to 2.5% (e.g., 0.8 to 1.7%, 0.9 to 1.5%, or 1.0 to 1.5%).

In some embodiments, an oil has an ALA content of 0.5 to 1.5%, an oleic acid content of 60 to 70% (e.g., 62 to 68%, 63 to 67%, or 65 to 66%), and a total saturated fatty acid content of 5 to 10%. In some embodiments, an oil has an ALA content of 0.6 to 1.5% (e.g., 0.7 to 1.4%, 0.8 to 1.3%, or 0.9 to 1.2%) and an oleic acid content of 71 to 80% (e.g., 72 to 78%, 72 to 76%, 73 to 75%, 74 to 77%, 74 to 78%, or 75 to 80%). The total saturated content of such an oil can be 3 to 8% (e.g., 4 to 6%, 4 to 5.5%, 4 to 5%, 5 to 7%, 6 to 8%, or 7 to 8%). In some embodiments, a canola oil can have an ALA content of 0.5 to 1.5%, an oleic acid content of 85 to 87% (e.g., 86 to 87%), and a total saturated fatty acid content of 5 to 6%. In some embodiments, an oil has an ALA content of 0.5 to 1.5%, an oleic acid content of 81 to 90% (e.g., 82 to 88% or 83 to 87%) oleic acid and a total saturated fatty acid content of 3.5 to 4.5% (e.g., 3.75 to 4.25%, 3.9 to 4.1%, or 4.0%).

Oils described herein can have an eicosenoic acid content of 1.0 to 1.9%. For example, an oil can have an eicosenoic acid content of 1.0 to 1.4%, 1.1 to 1.3%, 1.1 to 1.6%, 1.2 to 1.6%, 1.4 to 1.9%, in addition to a low ALA content.

Oils described herein can have a linoleic acid content of 3.5 to 26%, e.g., 3.7 to 4.5%, 8 to 10%, 9 to 12%, 10 to 13%, 11 to 13%, 12 to 16%, 13 to 16%, 14 to 18%, or 14 to 22%, in addition to a low ALA content.

Oils described herein have an erucic acid content of less than 2% (e.g., less than 1%, 0.5%, 0.2, or 0.1%) in addition to a low ALA content.

The fatty acid composition of seeds can be determined by first crushing and extracting oil from seed samples (e.g., bulk seeds samples of 10 or more seeds). TAGs in the seed are hydrolyzed to produce free fatty acids, which then can be converted to fatty acid methyl esters and analyzed using techniques known to the skilled artisan, e.g., gas-liquid chromatography (GLC) according to AOCS Procedure Ce 1e-91. Near infrared (NIR) analysis can be performed on whole seed according to AOCS Procedure Am-192 (revised 1999)

Seeds harvested from plants described herein can be used to make a crude canola oil or a refined, bleached, and deodorized (RBD) canola oil with a low ALA content. Harvested canola seed can be crushed to extract crude oil and, if desired, refined, bleached and deodorized by techniques known in the art. See, e.g., Bailey's Industrial Oil and Fat Products, Volume 5, “Edible Fat and Oil Products: Processing Technologies” (6th Edition, 2005). Briefly, refining refers to removing most if not all free fatty acids and other impurities such as phosphatides or protein substances from a crude oil. One common method of refining involves treating an oil with a strong base, followed by extensive washings with water. Bleaching refers to a process that removes natural pigments (e.g., carotenoids, chlorophylls, and xanthophylls) and other impurities such as metal cations (e.g., Fe, Cu and Zn). Bleaching can be done by absorbing such pigments and/or cations on a natural bleaching earth or clay, which is usually added to an oil under vacuum and high temperature. Deodorizing refers to the removal of relatively volatile trace components (e.g., ketones, aldehydes, alcohols) from an oil that contribute to flavor, odor, and color. Deodorizing is usually done by injecting steam into an oil heated to high temperatures (e.g., about 470° F. to about 510° F.) under high vacuum (e.g., <5 mm Hg).

In one useful example, the seed can be tempered by spraying the seed with water to raise the moisture to, for example, 8.5%. The tempered seed can be flaked using a smooth roller with, for example, a gap setting of 0.23 to 0.27 mm. Heat may be applied to the flakes to deactivate enzymes, facilitate further cell rupturing, coalesce the oil droplets, or agglomerate protein particles in order to ease the extraction process. Typically, oil is removed from the heated canola flakes by a screw press to press out a major fraction of the oil from the flakes. The resulting press cake contains some residual oil.

Crude oil produced from the pressing operation typically is passed through a settling tank with a slotted wire drainage top to remove the solids expressed out with the oil in the screw pressing operation. The clarified oil can be passed through a plate and frame filter to remove the remaining fine solid particles. Further oil can be extracted from the press cake produced from the screw pressing operation using known solvent extraction techniques, e.g., using commercial n-hexane extraction. The canola oil recovered from the solvent extraction process is combined with the clarified oil from the screw pressing operation, resulting in a blended crude oil.

Free fatty acids and gums typically are removed from the crude oil by heating in a batch refining tank to which food grade phosphoric acid has been added. The acid serves to convert the non-hydratable phosphatides to a hydratable form, and to chelate minor metals that are present in the crude oil. The phosphatides and the metal salts are removed from the oil along with the soapstock. The oil-acid mixture is treated with sodium hydroxide solution to neutralize the free fatty acids and the phosphoric acid in the acid-oil mixture. The neutralized free fatty acids, phosphatides, etc. (soapstock) are drained off from the neutralized oil. A water wash may be done to further reduce the soap content of the oil. The oil may be bleached and deodorized before use, if desired, by techniques known in the art.

Oils obtained from plants described herein can have increased oxidative stability, which can be measured using, for example, an Oxidative Stability Index Instrument (e.g., from Omnion, Inc., Rockland, Mass.) according to AOCS Official Method Cd 12b-92 (revised 1993). Oxidative stability is often expressed in terms of “AOM” hours.

Oils obtained from plants described herein also can have increased flavor stability, which can be measured using, for example, trained test panels in room-odor tests according to Mounts, J. Am. Oil Chem. Soc. 56:659-663, 1979 and the AOCS Recommended Practice Cg 2-83 for the Flavor Evaluation of Vegetable Oils (Methods and Standard Practices of the AOCS, 4th Edition (1989)). The technique encompasses standard sample preparation and presentation, as well as reference standards and method for scoring oils.

Food Compositions

This document also features food compositions containing the oils described above. For example, oils having a low ALA content (e.g., 0.5 to 1.5%) can be used for food applications or for frying. Oils having a low ALA content in combination with a low (6% or less) or very low (3.5% or less) total saturated fatty acid content can be used to replace or reduce the amount of saturated fatty acids and hydrogenated oils (e.g., partially hydrogenated oils) in various food products such that the levels of saturated fatty acids and trans fatty acids are reduced in the food products. In particular, canola oils having a low ALA content in combination with a low total saturated fatty acid content and a mid or high oleic acid content can be used to replace or reduce the amount of saturated fats and partially hydrogenated oils in processed or packaged food products, including bakery products such as cookies, muffins, doughnuts, pastries (e.g., toaster pastries), pie fillings, pie crusts, pizza crusts, frostings, breads, biscuits, and cakes, breakfast cereals, breakfast bars, puddings, and crackers.

For example, an oil described herein can be used to produce sandwich cookies that contain no or reduced levels of partially hydrogenated oils in the cookie and/or crème filling. In some embodiments, the cookies also have a reduced total saturated fatty acid content. Such cookie compositions can include, for example, in addition to canola oil, flour, sweetener (e.g., sugar, molasses, honey, high fructose corn syrup, artificial sweetener such as sucralose, saccharine, aspartame, or acesulfame potassium, and combinations thereof), eggs, salt, flavorants (e.g., chocolate, vanilla, or lemon), a leavening agent (e.g., sodium bicarbonate or other baking acid such as monocalcium phosphate monohydrate, sodium aluminum sulfate, sodium acid pyrophosphate, sodium aluminum phosphate, dicalcium phosphate, glucano-deltalactone, or potassium hydrogen tartrate, or combinations thereof), and optionally, an emulsifier (e.g., mono- and diglycerides of fatty acids, propylene glycol mono- and di-esters of fatty acids, glycerol-lactose esters of fatty acids, ethoxylated or succinylated mono- and diglycerides, lecithin, diacetyl tartaric acid esters or mono- and diglycerides, sucrose esters of glycerol, and combinations thereof). A crème filling composition can include, in addition to canola oil, sweetener (e.g., powdered sugar, granulated sugar, honey, high fructose corn syrup, artificial sweetener, or combinations thereof), flavorant (e.g., vanilla, chocolate, or lemon), salt, and, optionally, emulsifier.

Canola oils (e.g., with a low ALA, low total saturated fatty acid and low or high oleic acid content) also are useful for frying applications due to the polyunsaturated content, which is low enough to have improved oxidative stability for frying yet high enough to impart the desired fried flavor to the food being fried. For example, canola oils can be used to produce fried foods such as snack chips (e.g., corn or potato chips), French fries, or other quick serve foods.

Oils described herein also can be used to formulate spray coatings for food products (e.g., cereals or snacks such as crackers). In some embodiments, the spray coating can include other vegetable oils such as sunflower, cottonseed, corn, or soybean oils. A spray coating also can include an antioxidant and/or a seasoning.

Oils described herein also can be use in the manufacturing of dressings, mayonnaises, and sauces to provide a reduction in the total saturated fat content of the product. Oils described herein can be used as a base oil for creating structured fat solutions such as microwave popcorn solid fats or canola butter formulations.

Plant Breeding

The nucleic acids described herein (e.g., fad3D and fad3E nucleic acids) can be used as markers in plant genetic mapping and plant breeding programs. Such markers may include restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA detection (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) or microsatellite, for example. Marker-assisted breeding techniques may be used to identify and follow a desired fatty acid composition (e.g., low linolenic acid) during the breeding process. For example, a nucleic acid described herein, such as the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:32, or the complement thereof, can be used to identify one or more individual plants that possess the polymorphic allele correlated with the desired linolenic acid content. Those plants then can be used in a breeding program to combine the polymorphic allele with a plurality of other alleles at other loci that are correlated with a desired variation (e.g., in fatty acid composition). In some embodiments, a fragment of the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:32, or the complement thereof, that is at least 50 nucleotides in length can be used to distinguish a modified fad3 allele from a wild-type Fad3 allele (e.g., by allele-specific hybridization or by PCR).

Techniques suitable for use in a plant breeding program are known in the art and include, without limitation, backcrossing, mass selection, pedigree breeding, bulk selection, crossing to another population and recurrent selection. These techniques can be used alone or in combination with one or more other techniques in a breeding program. Thus, each identified plant is selfed or crossed to a different plant to produce seed, which is then germinated to form progeny plants. At least one such progeny plant is then selfed or crossed with a different plant to form a subsequent progeny generation. The breeding program can repeat the steps of selfing or outcrossing for an additional 0 to 5 generations as appropriate in order to achieve the desired uniformity and stability in the resulting plant line, which retains the polymorphic allele. In most breeding programs, analysis for the particular polymorphic allele will be carried out in each generation, although analysis can be carried out in alternate generations if desired.

In some cases, selection for other useful traits is also carried out, e.g., selection for disease resistance. Selection for such other traits can be carried out before, during or after identification of individual plants that possess the desired polymorphic allele.

Marker-assisted breeding techniques may be used in addition to, or as an alternative to, other sorts of identification techniques. An example of marker-assisted breeding is the use of PCR primers that specifically amplify a sequence containing a desired mutation in the fad3D or fad3E sequence.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

In the Tables described herein, the fatty acids are referred to by the length of the carbon chain and number of double bonds within the chain. For example, C14:0 refers to myristic acid; C16:0 refers to palmitic acid; C18:0 refers to stearic acid; C18:1 refers to oleic acid; C18:2 refers to linoleic acid; C18:3 refers to ALA; C20:0 refers to archidic acid; C20:1 refers to eicosenoic acid; C22:0 refers to behenic acid; C22:1 refers to erucic acid; C24:0 refers to lignoceric acid; and C24:1 refers to nervonic acid. “Total Sats” refers to the total of C14:0, C16:0, C18:0, C20:0, C22:0, and C24:0. Representative fatty acid profiles are provided for each of the specified samples.

Unless otherwise indicated, all percentages refer to wt % based on total wt % of fatty acids (i.e., fatty acid moieties) in the oil as determined by measuring the FAME moieties in accordance with the modified version of ROCS Ce 1c-89 set forth in Example 1.

Example 1 Brassica Plant Lines 1904 and 2558

Plants producing an oil with a low ALA content were obtained by subjecting a population of B. napus IMC201 seeds to chemical mutagenesis and selecting for low linolenic acid content (<1.5%). The typical fatty acid composition of field grown IMC201 is 3.6% C16:0, 1.8% C18:0, 76% C18:1, 12.5% C18:2, 3% C18:3, 0.7% C20:0, 1.5% C20:1, 0.3% C22:0, 0% C22:1, with total saturates of 6.4%. Prior to mutagenesis, IMC201 seeds were pre-imbibed in 700 gm seed lots by soaking for 15 min then draining for 5 min at room temperature. This was repeated four times to soften the seed coat. The pre-imbibed seed then were treated with 4 mM methyl N-nitrosoguanidine (MNNG) for three hours. Following the treatment with MNNG, seeds were drained of the mutagen and rinsed with water for one hour. After removing the water, the seeds were treated with 52.5 mM ethyl methanesulfonate (EMS) for sixteen hours. Following the treatment with EMS, the seeds were drained of mutagen and rinsed with water for one and one half hours. This dual mutagen treatment produced an LD₅₀ with the seed population.

Lines 1904 and 2558 were selected from the mutagenized population of IMC201 seeds as follows. Three thousand bulk M2 generation seeds were planted. Upon maturity, M3 seed (2500 individuals) was harvested from 2500 M2 plants and analyzed via a modified method for gas chromatograph determination of fatty acid profile per the American Oil Chemist's Society protocol (AOCS Ce 1c-89). In accordance with AOCS Ce 1c-89, the oil from the seeds was first treated to convert the acylglycerols to fatty acid methyl esters (“FAMEs”) and vials of the FAMEs were placed in a gas chromatograph for analysis in accordance with a modified version of American Oil Chemist's Society Official Method Ce 1-62 that employed an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, Calif.) equipped with a fused silica capillary column (5 m×0.180 mm and 0.20 μm film thickness) packed with a polyethylene glycol based DB-Wax® for liquid phase separation (J&W Scientific, Folsom, Calif.). Hydrogen (H2) was used as the carrier gas at a flow rate of 2.5 mL/min and the column temperature was isothermal at 200° C. Seed from each plant was tested via this method in replicates of two.

Lines 1904 and 2558 were identified as having low linolenic acid content in seed oil. M3 seeds of lines 1904 and 2558 were planted (50 per line) and the resulting plants were self pollinated. M4 seeds were harvested from the plants and bulk seed samples (approximately 20 seeds) were analyzed via GC. The results are presented in Table I Lines 1904 and 2558 had ALA contents ranging from approximately 0.70% to 1.95%. Line 1904 was deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-11273 and line 2558 was deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-11274.

TABLE 1 Fatty acid profile of harvested M4 generation mutant seed. RESCHID C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 M3B-1904-01 0.065 4.675 0.323 2.307 72.509 16.181 0.912 0.818 M3B-1904-02 0.052 4.183 0.256 2.507 74.995 13.520 1.431 0.911 M3B-1904-03 0.055 3.938 0.216 2.272 75.548 13.762 1.239 0.834 M3B-1904-04 0.058 4.089 0.218 2.313 74.369 14.354 1.507 0.862 M3B-1904-05 0.056 3.930 0.202 2.571 75.823 13.172 1.377 0.889 M3B-1904-06 0.062 4.286 0.256 2.170 74.700 14.110 1.195 0.829 M3B-1904-07 0.053 4.147 0.247 2.386 75.233 13.305 1.306 0.866 M3B-1904-08 0.058 4.198 0.237 2.530 74.591 13.613 1.217 0.965 M3B-1904-09 0.063 4.133 0.210 2.246 75.223 13.916 1.147 0.845 M3B-1904-10 0.068 4.367 0.300 2.708 71.414 16.504 1.386 0.891 M3B-1904-11 0.057 4.022 0.246 2.343 75.222 13.434 1.450 0.835 M3B-1904-12 0.058 4.157 0.247 2.315 74.670 14.226 1.160 0.816 M3B-1904-13 0.055 4.179 0.251 2.473 73.852 15.023 0.839 0.853 M3B-1904-14 0.059 4.151 0.251 2.268 75.153 13.876 1.107 0.819 M3B-1904-15 0.073 4.095 0.272 2.390 75.613 13.511 1.214 0.829 M3B-1904-16 0.054 3.947 0.210 2.653 75.732 13.173 1.516 0.869 M3B-1904-17 0.051 3.877 0.236 2.634 75.262 13.495 0.809 0.942 M3B-1904-18 0.057 3.901 0.240 2.598 75.561 13.259 1.268 0.908 M3B-1904-19 0.058 3.942 0.215 2.270 76.923 12.821 0.779 0.786 M3B-1904-20 0.066 4.044 0.263 2.105 74.938 14.222 1.436 0.758 M3B-1904-21 0.071 4.264 0.275 2.275 74.864 14.105 1.123 0.852 M3B-1904-22 0.060 4.242 0.257 2.308 75.173 13.872 1.137 0.817 M3B-1904-23 0.060 4.095 0.240 2.122 73.657 15.742 0.780 0.814 M3B-1904-24 0.068 4.046 0.272 2.294 74.481 14.038 1.251 0.848 M3B-1904-25 0.063 4.162 0.265 2.364 75.169 14.022 1.126 0.825 M3B-1904-26 0.054 3.981 0.246 2.228 76.741 13.190 0.741 0.795 M3B-1904-27 0.057 4.058 0.240 2.112 75.577 14.077 0.844 0.824 M3B-1904-28 0.058 4.221 0.274 2.259 74.558 13.776 1.219 0.837 M3B-1904-29 0.058 4.364 0.223 2.550 74.320 14.035 1.298 0.941 M3B-1904-30 0.054 4.133 0.225 2.419 74.744 13.928 1.317 0.926 M3B-1904-31 0.070 4.209 0.259 2.225 76.060 13.424 1.118 0.755 M3B-1904-32 0.079 4.867 0.385 3.113 69.744 16.279 1.951 1.048 M3B-1904-33 0.066 4.346 0.261 2.741 74.104 13.778 1.582 0.882 M3B-1904-34 0.079 4.571 0.269 2.605 73.843 14.412 1.272 0.889 M3B-1904-35 0.067 4.168 0.259 2.460 75.821 13.653 0.744 0.808 M3B-1904-36 0.051 4.128 0.209 2.338 76.117 12.715 1.416 0.894 M3B-1904-37 0.054 4.238 0.201 2.295 74.756 13.803 1.531 0.891 M3B-1904-38 0.057 4.325 0.237 2.472 75.481 13.396 1.460 0.825 M3B-1904-39 0.059 4.178 0.249 2.392 74.176 14.528 1.360 0.890 M3B-1904-40 0.056 4.176 0.245 3.409 72.309 13.105 1.378 1.171 M3B-1904-41 0.057 4.141 0.239 2.392 75.487 13.894 1.077 0.841 M3B-1904-42 0.054 3.947 0.225 2.488 74.792 14.490 1.131 0.831 M3B-1904-43 0.051 3.985 0.226 2.263 75.686 13.277 1.570 0.823 M3B-1904-44 0.052 4.137 0.202 2.677 75.733 12.649 1.506 0.950 M3B-1904-45 0.052 3.929 0.195 2.280 75.101 14.334 1.117 0.836 M3B-1904-46 0.060 4.354 0.283 2.577 73.385 13.857 1.635 0.975 M3B-1904-47 0.062 4.373 0.291 2.473 74.242 13.602 1.288 0.870 M3B-1904-48 0.054 4.074 0.233 2.174 75.247 13.779 1.143 0.830 M3B-1904-49 0.059 4.125 0.253 2.093 74.920 14.379 0.909 0.804 M3B-1904-50 0.060 4.157 0.235 2.037 74.522 14.402 1.539 0.786 M3B-2558-01 0.067 3.772 0.244 3.251 78.153 10.081 0.785 1.208 M3B-2558-02 0.058 3.674 0.218 2.989 78.233 10.027 1.181 1.095 M3B-2558-03 0.062 4.123 0.308 3.329 76.058 11.181 1.293 1.209 M3B-2558-04 0.065 4.134 0.295 2.915 75.964 12.077 0.974 1.029 M3B-2558-05 0.058 4.108 0.283 3.267 77.220 10.413 1.176 1.160 M3B-2558-06 0.064 3.801 0.254 3.057 78.311 9.731 1.076 1.094 M3B-2558-07 0.048 3.660 0.231 2.788 76.319 12.244 1.262 1.023 M3B-2558-08 0.069 3.845 0.275 3.494 76.433 10.690 1.309 1.271 M3B-2558-09 0.055 3.852 0.281 2.987 77.339 10.931 1.184 0.988 M3B-2558-10 0.055 3.864 0.267 3.079 77.765 9.847 1.509 1.103 M3B-2558-11 0.056 3.833 0.261 3.043 78.849 9.032 0.933 1.164 M3B-2558-12 0.049 3.996 0.218 2.955 77.218 11.409 0.814 1.056 M3B-2558-13 0.057 3.899 0.246 3.409 76.902 10.934 1.205 1.135 M3B-2558-14 0.056 3.984 0.223 3.775 78.095 8.899 1.184 1.323 M3B-2558-15 0.052 3.762 0.231 3.088 77.908 10.410 1.186 1.078 M3B-2558-16 0.053 3.865 0.245 3.360 77.868 9.754 1.223 1.176 M3B-2558-17 0.056 4.070 0.244 3.064 78.090 10.426 1.190 1.061 M3B-2558-18 0.073 4.064 0.259 2.869 76.821 11.402 0.878 1.053 M3B-2558-19 0.062 3.835 0.250 3.165 76.790 11.196 1.159 1.143 M3B-2558-20 0.071 3.987 0.289 3.482 76.133 10.992 1.238 1.240 M3B-2558-21 0.072 4.100 0.341 3.147 76.942 10.636 1.212 1.111 M3B-2558-22 0.055 3.875 0.279 2.700 76.780 11.604 1.486 0.930 M3B-2558-23 0.063 4.113 0.266 3.179 76.201 11.527 0.962 1.128 M3B-2558-24 0.058 3.779 0.252 2.895 77.381 10.867 1.161 1.035 M3B-2558-25 0.054 4.008 0.266 3.123 76.541 11.413 1.142 1.115 M3B-2558-26 0.063 3.956 0.283 3.197 76.084 11.172 1.262 1.166 M3B-2558-27 0.057 4.003 0.297 2.820 74.978 13.117 1.192 0.989 M3B-2558-28 0.061 3.837 0.277 3.584 75.859 11.272 1.217 1.235 M3B-2558-29 0.056 3.879 0.275 2.685 75.620 12.874 1.307 0.977 M3B-2558-31 0.059 3.933 0.266 2.919 76.965 11.336 0.918 1.097 M3B-2558-32 0.059 3.876 0.287 3.005 76.378 11.972 0.854 1.017 M3B-2558-33 0.060 4.204 0.271 3.251 74.489 12.869 1.378 1.132 M3B-2558-34 0.089 4.307 0.314 3.290 73.974 13.250 1.204 1.106 M3B-2558-35 0.053 3.747 0.208 3.144 77.721 10.975 0.722 1.128 M3B-2558-36 0.056 4.071 0.281 2.911 76.432 11.760 1.244 1.027 M3B-2558-37 0.053 3.931 0.265 3.287 76.237 11.366 1.446 1.141 M3B-2558-39 0.067 3.998 0.303 3.160 75.020 12.561 1.320 1.066 M3B-2558-40 0.051 3.895 0.251 3.493 76.324 10.978 1.476 1.190 M3B-2558-41 0.068 3.709 0.257 3.452 76.017 11.107 1.491 1.183 M3B-2558-42 0.062 3.932 0.258 3.214 76.168 11.541 1.138 1.094 M3B-2558-43 0.054 3.846 0.248 3.215 75.801 12.266 1.135 1.090 M3B-2558-44 0.051 3.782 0.279 3.271 76.059 11.827 1.487 1.082 M3B-2558-45 0.060 3.787 0.252 3.053 76.253 11.901 1.163 1.007 M3B-2558-46 0.054 3.758 0.255 3.377 78.569 9.676 0.844 1.183 M3B-2558-47 0.060 3.981 0.277 3.142 76.833 11.297 0.823 1.082 M3B-2558-48 0.052 3.816 0.264 3.204 76.778 11.235 1.305 1.112 M3B-2558-49 0.053 4.068 0.277 3.172 75.712 11.904 1.645 1.054 M3B-2558-50 0.059 4.063 0.282 3.440 75.763 11.199 1.607 1.126 TOT RESCHID C20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS M3B-1904-01 1.068 0.046 0.410 0.000 0.210 0.475 8.486 M3B-1904-02 1.180 0.046 0.453 0.000 0.267 0.201 8.372 M3B-1904-03 1.284 0.048 0.421 0.000 0.250 0.133 7.771 M3B-1904-04 1.234 0.052 0.437 0.017 0.243 0.247 8.003 M3B-1904-05 1.133 0.044 0.407 0.000 0.210 0.186 8.063 M3B-1904-06 1.206 0.050 0.463 0.000 0.258 0.415 8.068 M3B-1904-07 1.259 0.047 0.443 0.000 0.219 0.491 8.113 M3B-1904-08 1.315 0.052 0.526 0.031 0.307 0.359 8.584 M3B-1904-09 1.215 0.048 0.436 0.021 0.237 0.260 7.959 M3B-1904-10 1.190 0.060 0.443 0.000 0.304 0.364 8.780 M3B-1904-11 1.228 0.045 0.418 0.000 0.220 0.481 7.895 M3B-1904-12 1.215 0.045 0.395 0.000 0.198 0.498 7.939 M3B-1904-13 1.227 0.051 0.418 0.000 0.238 0.540 8.217 M3B-1904-14 1.190 0.049 0.433 0.000 0.249 0.394 7.979 M3B-1904-15 1.144 0.042 0.389 0.000 0.207 0.222 7.983 M3B-1904-16 1.146 0.043 0.374 0.000 0.181 0.104 8.077 M3B-1904-17 1.277 0.051 0.480 0.000 0.266 0.621 8.250 M3B-1904-18 1.190 0.046 0.434 0.000 0.228 0.311 8.126 M3B-1904-19 1.153 0.046 0.389 0.000 0.198 0.423 7.642 M3B-1904-20 1.215 0.044 0.373 0.000 0.217 0.320 7.561 M3B-1904-21 1.193 0.044 0.424 0.000 0.245 0.265 8.131 M3B-1904-22 1.130 0.045 0.392 0.000 0.208 0.361 8.025 M3B-1904-23 1.360 0.059 0.451 0.000 0.284 0.336 7.825 M3B-1904-24 1.372 0.048 0.468 0.036 0.238 0.541 7.962 M3B-1904-25 1.134 0.042 0.384 0.000 0.208 0.235 8.007 M3B-1904-26 1.133 0.042 0.398 0.000 0.193 0.259 7.648 M3B-1904-27 1.311 0.050 0.466 0.024 0.233 0.127 7.750 M3B-1904-28 1.348 0.049 0.458 0.000 0.230 0.713 8.063 M3B-1904-29 1.236 0.051 0.484 0.000 0.289 0.150 8.687 M3B-1904-30 1.296 0.054 0.501 0.000 0.264 0.140 8.297 M3B-1904-31 1.010 0.043 0.348 0.000 0.165 0.316 7.771 M3B-1904-32 1.272 0.066 0.548 0.000 0.350 0.300 10.005 M3B-1904-33 1.079 0.049 0.405 0.000 0.253 0.455 8.693 M3B-1904-34 1.063 0.050 0.430 0.000 0.197 0.319 8.771 M3B-1904-35 1.069 0.040 0.356 0.000 0.171 0.386 8.029 M3B-1904-36 1.240 0.048 0.467 0.000 0.245 0.132 8.122 M3B-1904-37 1.301 0.049 0.471 0.000 0.266 0.145 8.215 M3B-1904-38 1.062 0.045 0.357 0.000 0.181 0.103 8.216 M3B-1904-39 1.257 0.047 0.456 0.000 0.288 0.121 8.262 M3B-1904-40 1.309 0.054 0.610 0.000 0.400 1.777 9.823 M3B-1904-41 1.121 0.044 0.410 0.000 0.195 0.103 8.036 M3B-1904-42 1.242 0.046 0.388 0.000 0.220 0.145 7.928 M3B-1904-43 1.298 0.046 0.425 0.000 0.203 0.146 7.751 M3B-1904-44 1.217 0.046 0.456 0.000 0.221 0.155 8.493 M3B-1904-45 1.307 0.047 0.445 0.000 0.225 0.133 7.767 M3B-1904-46 1.233 0.053 0.550 0.000 0.317 0.721 8.833 M3B-1904-47 1.232 0.047 0.451 0.000 0.246 0.824 8.475 M3B-1904-48 1.333 0.051 0.454 0.020 0.266 0.343 7.851 M3B-1904-49 1.281 0.057 0.457 0.000 0.248 0.416 7.786 M3B-1904-50 1.209 0.053 0.425 0.015 0.233 0.329 7.698 M3B-2558-01 1.313 0.037 0.583 0.016 0.354 0.137 9.234 M3B-2558-02 1.326 0.045 0.547 0.016 0.305 0.288 8.668 M3B-2558-03 1.278 0.045 0.604 0.000 0.362 0.150 9.689 M3B-2558-04 1.325 0.046 0.509 0.000 0.304 0.364 8.956 M3B-2558-05 1.259 0.044 0.545 0.000 0.332 0.136 9.469 M3B-2558-06 1.302 0.039 0.523 0.000 0.292 0.456 8.831 M3B-2558-07 1.394 0.059 0.511 0.000 0.254 0.208 8.283 M3B-2558-08 1.372 0.044 0.643 0.000 0.374 0.181 9.696 M3B-2558-09 1.206 0.038 0.442 0.000 0.232 0.464 8.556 M3B-2558-10 1.261 0.041 0.539 0.000 0.303 0.369 8.942 M3B-2558-11 1.377 0.040 0.618 0.000 0.388 0.408 9.102 M3B-2558-12 1.282 0.047 0.516 0.000 0.300 0.140 8.872 M3B-2558-13 1.254 0.044 0.523 0.000 0.297 0.094 9.321 M3B-2558-14 1.297 0.040 0.637 0.000 0.350 0.137 10.125 M3B-2558-15 1.305 0.045 0.521 0.000 0.297 0.117 8.797 M3B-2558-16 1.231 0.038 0.545 0.000 0.329 0.313 9.328 M3B-2558-17 1.284 0.000 0.516 0.000 0.000 0.000 8.767 M3B-2558-18 1.301 0.041 0.530 0.000 0.276 0.433 8.866 M3B-2558-19 1.325 0.043 0.546 0.000 0.314 0.172 9.065 M3B-2558-20 1.328 0.042 0.593 0.000 0.384 0.220 9.758 M3B-2558-21 1.247 0.040 0.537 0.000 0.340 0.276 9.306 M3B-2558-22 1.214 0.039 0.427 0.000 0.215 0.396 8.201 M3B-2558-23 1.313 0.045 0.576 0.000 0.340 0.287 9.399 M3B-2558-24 1.302 0.045 0.514 0.023 0.307 0.381 8.588 M3B-2558-25 1.302 0.046 0.535 0.017 0.304 0.134 9.140 M3B-2558-26 1.310 0.047 0.607 0.017 0.363 0.474 9.351 M3B-2558-27 1.321 0.049 0.506 0.027 0.288 0.356 8.663 M3B-2558-28 1.294 0.044 0.596 0.025 0.353 0.348 9.666 M3B-2558-29 1.327 0.056 0.510 0.000 0.288 0.147 8.394 M3B-2558-31 1.354 0.048 0.583 0.000 0.367 0.155 8.958 M3B-2558-32 1.250 0.042 0.490 0.000 0.273 0.499 8.719 M3B-2558-33 1.289 0.054 0.564 0.000 0.301 0.138 9.513 M3B-2558-34 1.239 0.047 0.518 0.000 0.284 0.379 9.593 M3B-2558-35 1.312 0.042 0.532 0.000 0.296 0.121 8.899 M3B-2558-36 1.287 0.050 0.523 0.000 0.243 0.115 8.831 M3B-2558-37 1.281 0.045 0.519 0.000 0.306 0.124 9.237 M3B-2558-39 1.283 0.048 0.515 0.000 0.303 0.356 9.110 M3B-2558-40 1.281 0.046 0.557 0.000 0.335 0.125 9.520 M3B-2558-41 1.251 0.044 0.577 0.000 0.344 0.502 9.332 M3B-2558-42 1.247 0.044 0.525 0.000 0.284 0.493 9.111 M3B-2558-43 1.339 0.049 0.521 0.000 0.309 0.128 9.034 M3B-2558-44 1.226 0.049 0.492 0.000 0.277 0.120 8.954 M3B-2558-45 1.259 0.047 0.473 0.015 0.259 0.472 8.638 M3B-2558-46 1.269 0.036 0.551 0.000 0.318 0.110 9.241 M3B-2558-47 1.261 0.038 0.510 0.000 0.291 0.405 9.065 M3B-2558-48 1.282 0.045 0.531 0.000 0.265 0.111 8.980 M3B-2558-49 1.188 0.047 0.493 0.000 0.273 0.114 9.114 M3B-2558-50 1.215 0.042 0.503 0.000 0.272 0.431 9.462

Selected M4 individuals were self pollinated to generate M5 seeds and further evaluated in an environmentally controlled plant growth chamber. Seeds from M3B-2558-35 and M3-B1904-35 were planted in Premier Pro-Mix BX potting soil (Premier Horticulture, Quebec, Canada) in four inch plastic pots. Planted seeds were watered and stratified at 5° C. for 5 days and germinated at 20° C. day temperature and 17° C. night temperature (20/17) in Conviron ATC60 controlled-environment growth chambers (Controlled Environments Limited, Winnipeg, MB). Each genotype combination was randomized and replicated 10 times in each of two separate growth chambers. At flowering, one chamber was reduced to a diurnal temperature cycle of 15° C. day temperature and 12° C. night temperature (15/12) while the other remained at 20/17. The temperature treatments were imposed to identify the effects of temperature on fatty acid composition. Plants were watered five times per week and fertilized bi-weekly using a 20:20:20 (NPK) liquid fertilizer at a rate of 150 ppm. Plants were bagged individually to ensure self pollination and genetic purity of the seed. Seeds from each plant were harvested individually at physiological seed maturity. The fatty acid profile of the seeds was determined using the modified GC method described above (replicates of two).

Fatty acid data from plants grown under the different temperature regimes was analyzed in two ways. First, data was analyzed separately as different environments and then it was pooled and analyzed across environments. Data was analyzed in SAS (SAS Institute, 2003) using proc glm to estimate differences in mean fatty acid values. Table 2 contains the population size, mean value and standard deviation of oleic, linoleic and linolenic fatty acid of seeds produced by plants carrying mutant fad3 alleles and grown in two environmental growth chambers set at different diurnal temperature regimes (20° C. day/17° C. night; 15° C. day/12° C. night) as discussed above. Genotypes 1904-35 and 2558-35 are mutant allele combinations and v1030 hybrid and IMC02 are controls. The 1904-35, 2558-35, and IMC02 lines each contain mutant fad3A and fad3B alleles, while line 1904-35 also contains a mutant fad3E allele and a mutant fad3D allele (see below). Means with different letters are significantly different as determined by a Student-Newman-Keuls mean separation test. In conclusion, lines 1904-35 and 2558-35 can reach an alpha-linolenic content less than v1030 and IMC02.

Seeds of lines 1904 and 2558 were deposited with the American Type Culture Collection (ATCC) (Manassas, Va.) on Sep. 1, 2010, under conditions of the Budapest Treaty and assigned Accession Nos. PTA-11273 and PTA-11274, respectively. All restrictions upon public access to the deposits will be irrevocably removed upon grant of the patent. The deposits will be replaced if the depository cannot dispense viable samples.

TABLE 2 Mean oleic, linoleic and linolenic acid content in two environments Mean Mean Mean RESCHID C18:1 s.d. C18:2 s.d. C18:3 s.d. N 15/12 Environment v1030 65.877 0.564 22.031 0.523 3.430 a 0.116 9 IMC02 69.728 1.528 20.484 1.434 1.815 b 0.109 9 1904-35 73.986 1.437 16.956 1.369 1.071 c 0.082 10 2558-35 77.276 1.191 13.051 1.505 0.976 d 0.081 10 17/20 Environment v1030 65.053 1.397 22.906 1.570 2.952 a 0.133 10 IMC02 72.211 1.604 17.543 1.986 1.378 b 0.098 10 1904-35 77.009 0.475 13.477 0.489 1.052 c 0.040 9 2558-35 78.470 0.924 11.238 1.129 0.993 c 0.080 10 Across Environments V1030 65.443 1.138 22.491 1.247 3.179 a 0.274 19 IMC02 71.035 1.987 18.936 2.272 1.585 b 0.246 19 1904-35 75.418 1.881 15.308 2.056 1.062 c 0.065 19 2558-35 77.873 1.205 12.145 1.595 0.984 c 0.079 20

Example 2 Identification of a Fad3E Mutation in 1904-35 Plants

Genome mapping, map-based gene cloning, and direct-sequencing strategies were used to identify loci associated with the <1.5% linolenic fatty acid content in the 1904-35 line described in Example 1. A DH (doubled haploid) population was developed from a cross between 1904-35 and 95CB504, a B line (maintainer). The two parental lines were screened with 1066 SNP (single nucleotide polymorphism) markers using the MassARRAY platform (Sequenom Inc., San Diego, Calif.) to identify polymorphic SNP markers between the two parents; 174 polymorphic SNP markers were identified.

Single marker correlations and multiple regression analysis between fatty acid composition and SNP markers were carried out using the SAS program (SAS Institute 1988). A Brassica napus genetic linkage map was constructed using the Kosambi function in JoinMap 3.0 (Kyazma). Interval mapping for quantitative trait loci (QTL) was done with MapQTL 4.0 (Kyazma). A LOD score >3.0 was considered as the significance threshold to declare the association intervals.

Comparative genome mapping was performed to locate the identified QTL in Brassica napus chromosomes and further identify the Brassica rapa BAC (Bacterial Artificial Chromosome) clones encompassing the identified SNP markers and the candidate genes in the identified QTL interval for the <1.5% linolenic acid content using publicly available Brassica and Arabidosis genome sequences, genes, genetic linkage maps, and other information from the world wide web at brassica.bbsrc.ac.uk/, and ncbi.nlm.nih.gov/.

A total of 217 DH lines were genotyped with 174 polymorphic SNP markers. QTL mapping identified two QTLs for low linolenic acid content (<1.5% C18:3). Comparative genome mapping located one QTL on the N3 chromosome in Brassica napus (A3 in Brassica rapa) and further identified a Fad3E candidate gene which is located at 1cM from the SNP marker that showed significant association with C18:3 content. The 1cM interval between the SNP marker and Fad3E gene is 248 kb according to co-linearity with the Arabidopsis genome. Example 3 describes the second QTL on the N5 chromosome in Brassica napus (A5 in Brassica rapa).

The Fad3E genes from chromosome N3 of the Brassica napus genome were sequenced from 1904-35, 95CB504 and IMC201. The sequences were analyzed using BLAST (the Basic Local Alignment Search Tool) and DNASTAR/Lasergene 8.0 (DNASTAR, Inc). A single nucleotide substitution was identified in one of the two Fad3E isoforms from the 1904-35 mutant line that was not present in 95CB504 and IMC201. FIG. 1 shows the sequence alignment of the BnFad3E gene from 1904-35 and IMC201, and the BrFad3E located in Brassica rapa BAC, KBrH013B15 from the world wide web at brassica-rapa.org. The nucleotide substitution of a “A” in 1904-35 for “G” in IMC201 and 95CB504 at position 1851 of this alignment (position 1756 in SEQ ID:NO:1). As shown in FIG. 2, this transition mutation of Fad3E is at the exon 3, intron 3 border. FIG. 3 shows the alignment of FAD3E amino acid sequences from 1904 BnFAD3E-2 and IMC201 BnFAD3E-2 (SEQ ID NO:29), BrFAD3E deduced from BrFad3E (world wide web at brassica-rapa.org) (SEQ ID NO:30), and AtFAD3 (GenBank accession number: NP_(—)180559; SEQ ID NO:31). The fad3E-2 SNP allele results in an altered consensus sequence at the “splice donor site” for RNA splicing. Therefore, the RNA splicing of fad3E-2 primary transcript (pre-mRNA) cannot be processed to create a mature RNA (mRNA).

Large scale screening of the parental lines (1904-35 and 95CB504) as well as other Brassica napus cultivars including 2558, indicated the fad3E-2 SNP allele was 1904-35-specific and was significantly associated with the low ALA phenotype (R-square=0.275 for C18:3 content) using 217 DH lines developed from the cross between 1904-35 and 95CB504. This 1904-35 fad3E-2 SNP allele also was present in selections having <1.5% C18:3 content from a backcross population developed from the cross between 1904-35 and 1035R, an R line (restorer).

Example 3 Identification of a Fad3D Mutation in 1904-35 Plants

As indicated in Example 2, a 2^(nd) QTL was also identified for low linolenic acid content. Comparative genomics located this 2nd QTL on the N5 chromosome of Brassica napus and further identified a Fad3D candidate gene on chromosome N5. The Fad3D genes from chromosome N5 of the Brassica napus genome were sequenced from 1904-35, 95CB504, and IMC201. The sequences were analyzed using BLAST and DNASTAR/Lasergene 8.0 (DNASTAR, Inc). FIG. 4 shows the sequence alignment of a portion of the BnFad3D gene from 1904-35, 95CB504 and IMC201.

A deletion was identified in one of the two Fad3D isoforms from the 1904-35 mutant line that was not present in 95CB504 and IMC201. The mutant type BnFad3D from 1904-35 has a deletion including a portion of exon 1 (from position 575 to position 739). In IMC201 and 95CB504, exon 1 starts at position 441 and ends at position 739. As a result of the deletion in 1904-35, exon 1 is only 134 bp long. Therefore, it is believed the deletion mutation in 1904 BnFad3D induced a non-functional truncated protein/enzyme due to either lack of RNA splicing (truncated protein with 64 amino acids) or incorrect RNA splicing (truncated protein).

Large scale screening of the parental lines (1904-35 and 95CB504) as well as other Brassica napus cultivars, indicated the Fad3D deletion was 1904-35-specific. In addition, the Fad3D deletion was significantly associated with the low ALA phenotype (R-square=0.61, equal to 61% phenotypic variation on C18:3) using the parental lines and 77 DH lines developed from the cross between 1904-35 and 95CB504 compared with 22% explained by BnFad3E-2 mutation in 1904-35. In order to determine the relative effect of individual Fad3 isoform on C18:3 content, 215 lines were used from multiple populations, which carry all Fad3 isoforms, for the multiple regression analysis. Results demonstrated that BnFad3B explains the largest proportion of phenotypic variation on C18:3 content with 26%, followed by 16% by BnFad3D, 8% by BnFad3A, and 7% by BnFad3E.

Example 4 Mutant Fad3A and Fad3B Genes

A population of B. napus IMC201 seeds was subjected to chemical mutagenesis as set forth in Example 1. Approximately 200,000 treated seeds were planted in standard greenhouse potting soil and placed into environmentally controlled greenhouse. The plants were grown under sixteen hours of day light. At maturity, M2 seed was harvested from the plants and bulked together. The M2 generation was planted and leaf samples from the early, post-cotyledon stage of development from 8 plants were pooled and DNA was extracted from leaves of these plants. The leaf harvest, pooling and DNA extraction was repeated for approximately 32,000 plants, and resulted in approximately forty 96-well blocks containing mutagenized B. napus IMC201 DNA. This grouping of mutagenized DNA is referred to below as the original DNA mutagenesis library.

Additionally, approximately 200,000 treated seeds from the dual mutagen treatment described in Example 1 were planted in standard greenhouse potting soil and placed into environmentally controlled greenhouse. The plants were grown under sixteen hours of day light. At maturity, M2 seed was harvested from the plants and bulked together. This M2 generation was planted in greenhouses and, at flowering, plants were bagged in groups of four to facilitate cross-pollination that would occur in parallel with the majority self pollination events, and seed from this generation was harvested. Genomic DNA from three seeds per plant of this M3 generation was isolated in 96-well blocks; a collection of mutagenized DNA from this process is referred to below as the new Tilling DNA mutagenesis library.

The original DNA mutagenesis library and the new Tilling DNA mutagenesis library were screened to identify stop-codon containing fad3A and fad3B mutant alleles. PCR reactions were performed using B. napus IMC201 genomic DNA original mutagenesis library or new Tilling DNA mutagenesis library. PCR products from the original mutagenesis library were analyzed using temperature gradient capillary electrophoresis on a REVEAL® instrument (Transgenomics Inc.), which allows PCR reactions containing heterogeneous PCR products to be distinguished from reactions containing only homogeneous products, as would be the case if a SNP existed in genomic DNA from chemical mutagenesis and subsequent PCR amplification. The PCR products from the new Tilling DNA mutagenesis library were sequenced directly using an Applied Biosystems (Life Technologies) 3730 DNA sequencer using the manufacturer's recommendations.

Individual seeds representing the primary hit of each M2 plant that was the source genomic DNA mix for this primary mutagenesis screen were sampled and genomic DNA was isolated in order to perform the Fad3A PCR on these individuals. PCR products were sequenced and the sequences were compared to the wild-type sequence to screen for the presence of an induced stop codon.

The sequence comparisons indicated that a mutation had been generated and mutant plants obtained for each of the Fad3A and Fad3B genes. The mutant Fad3A sequence is shown in SEQ ID NO: 9 and contains a mutation at position 102, changing the codon from TGG to TGA. The mutant Fad3B sequence is shown in SEQ ID NO:10 and contains a mutation at position 206, resulting in a codon change from TGG to TAG.

Example 5 DH Line Husker

A cross was made between 1904-35 (Example 1) and 95CB504, a B line (maintainer). A double haploid population was generated by collecting F₁ microspores from the cross, treating the microspores with colchicine, and propagating them in vitro. Plantlets formed in vitro from the microspores were moved to a greenhouse and inflorescences that formed were self pollinated. Seed was harvested from the DH₁ plants at maturity and analyzed for fatty acid profile. Seeds from those plants exhibiting low and high linolenic acid content were grown in the greenhouse. Table 3 contains the fatty acid profile of a bulk sample of seeds produced by each of 5-10 greenhouse-grown plants of a DH₁ population designated Husker.

TABLE 3 Fatty acid profile of DH₁ population designated Husker RESCHID Pedigree n C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Husker- 95CB504xM3B- 5 0.00 2.308 0.078 1.728 80.758 11.130 0.766 0.774 100 1904-35 Husker- 95CB504xM3B- 3 0.00 3.353 0.140 2.310 78.293 11.920 0.923 0.887 141 1904-35 Husker- 95CB504xM3B- 5 0.03 3.336 0.160 2.048 78.186 11.984 0.858 0.938 147 1904-35 Husker- 95CB504xM3B- 5 0.03 3.688 0.242 2.246 78.816 10.864 0.814 0.920 161 1904-35 Husker- 95CB504xM3B- 5 0.04 4.130 0.256 2.070 75.004 12.408 2.444 0.934 107 1904-35 Husker- 95CB504xM3B- 5 0.02 3.363 0.175 2.080 78.211 11.661 1.161 0.891 125 1904-35 Husker- 95CB504xM3B- 5 0.02 3.574 0.195 2.151 77.702 11.767 1.240 0.914 138 1904-35 Husker- 95CB504xM3B- 4 0.04 4.768 0.268 1.808 75.962 11.738 2.336 0.780 170 1904-35 Husker- 95CB504xM3B- 5 0.01 2.765 0.153 1.900 79.270 12.533 0.738 0.718 314 1904-35 Husker- 95CB504xM3B- 5 0.03 4.526 0.168 2.370 75.004 11.948 2.338 0.934 323 1904-35 95CB504 9 0.05 3.723 0.231 2.518 78.586 9.073 2.256 1.030 TOT RESCHID Pedigree n C20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS Husker- 95CB504xM3B- 5 1.468 0.030 0.442 0.008 0.266 0.248 5.516 100 1904-35 Husker- 95CB504xM3B- 3 1.227 0.000 0.447 0.000 0.233 0.273 7.227 141 1904-35 Husker- 95CB504xM3B- 5 1.346 0.040 0.550 0.000 0.310 0.224 7.210 147 1904-35 Husker- 95CB504xM3B- 5 1.330 0.016 0.484 0.004 0.304 0.244 7.670 161 1904-35 Husker- 95CB504xM3B- 5 1.474 0.030 0.598 0.006 0.364 0.242 8.140 107 1904-35 Husker- 95CB504xM3B- 5 1.369 0.023 0.504 0.004 0.295 0.246 7.153 125 1904-35 Husker- 95CB504xM3B- 5 1.349 0.022 0.517 0.003 0.301 0.246 7.480 138 1904-35 Husker- 95CB504xM3B- 4 1.344 0.018 0.478 0.000 0.232 0.224 8.108 170 1904-35 Husker- 95CB504xM3B- 5 1.150 0.038 0.338 0.000 0.170 0.220 5.903 314 1904-35 Husker- 95CB504xM3B- 5 1.568 0.034 0.538 0.010 0.324 0.208 8.722 323 1904-35 95CB504 9 1.446 0.040 0.533 0.017 0.321 0.189 8.163

Example 6 DH Line Vest

A cross was made between 2558-35 (Example 1) and Dumpling-314, a double haploid B-line (maintainer) developed from a cross between IMC106RR and Jetton, a known winter rapeseed variety. A double haploid population was generated as described in Example 5. Seed was harvested from the DH₁ plants at maturity and analyzed for fatty acid profile. Seeds from those plants exhibiting low linolenic acid content were grown in the greenhouse. Table 4 contains the fatty acid profile of a bulk sample of seeds produced by each of 10 greenhouse-grown plants of a DH₁ population designated Vest.

TABLE 4 Mean fatty acid profile of Vest DH lines from tails of C18:3 distribution (Vest Population N = 51) Research ID Pedigree C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 Vest-70 Dump314-05xM3B- 0.069 4.507 0.223 1.983 67.152 22.456 0.653 0.778 2558-35-2 Vest-52 Dump314-05xM3B- 0.060 4.001 0.308 1.844 77.279 12.080 0.680 0.876 2558-35-2 Vest-86 Dump314-05xM3B- 0.065 4.406 0.264 2.266 79.352 9.008 0.745 1.009 2558-35-2 Vest-60 Dump314-05xM3B- 0.072 4.685 0.273 3.143 77.323 10.143 0.787 1.217 2558-35-2 Vest-87 Dump314-05xM3B- 0.082 5.083 0.346 2.317 68.253 18.677 0.790 1.001 2558-35-2 Vest-75 Dump314-05xM3B- 0.101 5.060 0.442 2.760 70.842 14.678 1.830 1.142 2558-35-2 Vest-69 Dump314-05xM3B- 0.143 5.840 0.653 3.079 65.526 18.353 1.952 1.359 2558-35-2 Vest-71 Dump314-05xM3B- 0.103 5.719 0.515 4.595 57.872 23.001 2.039 1.673 2558-35-2 Vest-92 Dump314-05xM3B- 0.137 6.678 0.606 3.026 54.170 28.786 2.423 1.300 2558-35-2 Vest-97 Dump314-05xM3B- 0.126 6.439 0.643 3.483 54.626 27.270 2.528 1.315 2558-35-2 Dumpling-314 avg 0.051 4.405 0.255 2.259 67.459 20.564 1.396 0.969 (n = 4) Research TOT ID Pedigree C20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS Vest-70 Dump314-05xM3B- 1.191 0.066 0.431 0.048 0.249 0.193 8.017 2558-35-2 Vest-52 Dump314-05xM3B- 1.562 0.053 0.571 0.043 0.411 0.233 7.762 2558-35-2 Vest-86 Dump314-05xM3B- 1.486 0.044 0.623 0.044 0.468 0.220 8.838 2558-35-2 Vest-60 Dump314-05xM3B- 1.287 0.042 0.597 0.000 0.430 0.000 10.145 2558-35-2 Vest-87 Dump314-05xM3B- 1.284 0.064 0.624 0.000 0.505 0.976 9.612 2558-35-2 Vest-75 Dump314-05xM3B- 1.415 0.067 0.657 0.000 0.516 0.491 10.235 2558-35-2 Vest-69 Dump314-05xM3B- 1.324 0.111 0.842 0.000 0.626 0.192 11.889 2558-35-2 Vest-71 Dump314-05xM3B- 1.125 0.085 1.005 0.000 0.660 1.608 13.755 2558-35-2 Vest-92 Dump314-05xM3B- 1.191 0.000 0.695 0.000 0.628 0.359 12.464 2558-35-2 Vest-97 Dump314-05xM3B- 1.088 0.000 0.712 0.441 0.616 0.715 12.690 2558-35-2 Dumpling-314 avg 1.380 0.062 0.571 0.016 0.454 0.160 8.708 (n = 4)

Example 7 Development of Hybrid Canola Producing Reduced ALA in the Seed Oil

A hybrid canola line yielding seeds with an ALA content of less than 1.5% was produced by introducing genes from line 1904-35 (Example 1) into a commercially grown hybrid variety, Victory® v1035. Hybrid v1035 has an average oleic acid content of 65% and an ALA content of 2.8%. Plants of the line 1904-35, and the inbreds 1035R and 95CB504, were planted in a greenhouse. Inbred 1035R is the male parent of v1035. Inbred 95CB504 is the B line female parent of v1035. Plants of 1035R and 1904-35 were cross pollinated in the greenhouse, as were 95CB504 and 1904-35, as shown in Table 5.

TABLE 5 Female x Male 1035R (R-line) 1904-35 95CB504 (B-line) 1904-35

F₁ progeny from the cross of 95CB504 and 1904-35 were backcrossed to 95CB504 to produce BC₁-B progeny, which were selfed (BC₁S). Plants with low total saturates were selected from the BC₁-B selfed progeny, and backcrossed to 95CB504 to produce BC₂-B progeny. F₁ progeny from the cross of 1035R and 1904-35 were backcrossed to 1035R to produce BC₁-R progeny, which were selfed. Plants with low linolenic were selected from the BC₁-R selfed progeny, and backcrossed to 1035R to produce BC₂-R progeny. Backcrossing, selection, and self-pollination of the BC-B and BC-R progeny were continued for multiple generations. The 95CB504 male sterile A line, 000A05 was converted to a low linolenic phenotype in parallel with the conversion of the 95CB504 B line. Table 6 shows the mean C18:3 content of selected lines of converted BC₃S₅ generation parent lines compared unconverted 95CB504 and 1035R.

Hybrid seed was generated by hand, using BC₁S₃ generation plants of the 95CB504 B line as the female parent and BC₁S₃ generation plants of the 1035R R line as the male parent. The hybrid seed was grown at 5 locations×4 replications in Western Canada. In the trial plot locations, some individual plants were bagged for self pollination (5 locations×2 reps) and seeds harvested at maturity. The remaining plants were not bagged (5 locations×4 reps) and seeds were harvested in bulk. As such, the bulk samples had some level of out crossing with non-low linolenic fatty acid lines in adjacent plots. Seeds from the individual and bulk samples were analyzed for fatty acid content. Seeds from control plants of line Q2, hybrid v1035 and commercial variety 46A65 were also harvested individually and in bulk.

Table 7 shows the fatty acid profile of the individually bagged samples and bulked samples for hybrid 1904-35 and controls. The results indicate that seed produced by Hybrid 1904-35 has a statistically significant decrease in 18:3 content relative to the controls.

TABLE 6 Mean linolenic acid content of parental controls and converted parents. ANOVA BC3S5 Populations Greenhouse ID Pedigree Mean C18:3 N 09AP:Waring42 a 1035R 1.914 8 09AP:Waring43 a 95CB504 1.892 6 09AP:Waring30 b 95CB504xM3B-1904 1.231 20 09AP:Waring25 b 95CB504xM3B-1904 1.226 20 09AP:Waring27 b 95CB504xM3B-1904 1.206 20 09AP:Waring28 b 95CB504xM3B-1904 1.198 20 09AP:Waring29 b 95CB504xM3B-1904 1.174 20 09AP:Waring32 b 95CB504xM3B-1904 1.165 20 09AP:Waring26 b 95CB504xM3B-1904 1.163 20 09AP:Waring33 b 95CB504xM3B-1904 1.146 20 09AP:Waring31 c 95CB504xM3B-1904 1.081 20 09AP:Waring7 d 1035R BxM3B-1904 0.797 20 09AP:Waring18 de 1035R BxM3B-1904 0.780 20 09AP:Waring14 def 1035R BxM3B-1904 0.773 20 09AP:Waring11 def 1035R BxM3B-1904 0.760 20 09AP:Waring13 def 1035R BxM3B-1904 0.755 18 09AP:Waring8 def 1035R BxM3B-1904 0.755 20 09AP:Waring16 def 1035R BxM3B-1904 0.752 20 09AP:Waring21 def 1035R BxM3B-1904 0.751 19 09AP:Waring17 def 1035R BxM3B-1904 0.751 20 09AP:Waring12 def 1035R BxM3B-1904 0.737 20 09AP:Waring19 def 1035R BxM3B-1904 0.728 20 09AP:Waring10 efg 1035R BxM3B-1904 0.692 20 09AP:Waring9 efg 1035R BxM3B-1904 0.691 20 09AP:Waring20 efg 1035R BxM3B-1904 0.681 20 09AP:Waring15 fg 1035R BxM3B-1904 0.668 20 09AP:Waring23 g 1035R BxM3B-1904 0.634 20 09AP:Waring22 g 1035R BxM3B-1904 0.632 19 09AP:Waring24 g 1035R BxM3B-1904 0.598 20 * Demonstrates significant difference between recurrent parent and backcross selections

TABLE 7 Mean linolenic fatty acid content of converted hybrid, v1035 and controls. reschid mean s.e. N Student-Newman-Keuls Tests for C18_3 Data Pooled for Bulked and Selfed Seed Q2 8.142 a 0.218 30 46A65 7.528 b 0.098 29 V1035 2.874 c 0.170 31 1904 Conversion 1.504 d 0.118 30 Data Separated for Bulked and Selfed Seed Q2 8.206 a 0.536 11 Q2 Bulk 8.105 a 0.166 19 46A65 7.823 a 0.168 10 46A65 Bulk 7.372 a 0.107 19 V1035 3.156 b 0.481 10 V1035 Bulk 2.739 b 0.108 21 1904 Conversion 1.546 c 0.123 19 Bulk 1904 Conversion 1.431 c 0.250 11

Example 8

Crosses were made between selections from lines of double haploid population Vest (Example 6) and reduced saturated fatty acid lines F6 (1764-43-06×1975-90-14) and 06JAXB (01OB054R×15.36). The reduced saturated fatty acid lines are described in U.S. Provisional Application No. 61/287,985, filed Dec. 18, 2009, and U.S. Provisional Application No. 61/295,049, filed Jan. 14, 2010. Double haploid populations were generated from these crosses as described in Example 4. Seed was harvested from the DH1 plants at maturity and analyzed for fatty acid profile. Table 8 contains the fatty acid profile of seeds produced by each of 1 greenhouse-grown plant from DH1 populations designated GP#1, GP#4 and GP#5 as well as 3 parental lines grown as reduced linolenic and total saturated fatty acid controls.

TABLE 8 Research ID Pedigree C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 GP #1- Vest-57x(1764-43-6x1975- 0.03 2.62 0.16 1.06 77.82 14.26 1.03 0.46 396 90-14) GP #1-24 Vest-57x(1764-43-6x1975- 0.05 3.11 0.29 0.95 74.63 16.92 1.06 0.47 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.04 3.11 0.26 1.02 75.24 16.41 1.06 0.46 181 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.03 2.78 0.23 1.15 76.61 14.89 0.99 0.53 444 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.03 3.02 0.25 1.11 77.22 14.22 1.08 0.51 449 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.00 2.80 0.20 1.35 74.47 16.93 1.04 0.55 240 90-14) GP #1- Vest-57x(1764-43-6x1975- 0.04 2.59 0.19 1.54 79.41 12.02 0.97 0.63 150 90-14) GP # 4- Vest-70x(1764-43-6x1975- 0.02 2.74 0.17 1.28 75.26 16.19 1.04 0.55 20 90-14) GP # 4- Vest-70x(1764-43-6x1975- 0.03 3.48 0.16 1.22 65.47 25.83 0.82 0.51 17 90-14) GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 2.83 0.13 1.56 77.51 13.35 1.09 0.66 434 57-05 GP # 5-* (01OB054RxLSAt15.36)xVest- 0.03 2.99 0.23 1.31 86.21 4.37 1.11 0.67 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.02 3.47 0.00 1.43 76.99 14.12 0.86 0.60 351 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 3.16 0.18 1.52 73.74 16.87 1.05 0.62 334 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.03 2.96 0.15 1.61 79.04 11.59 1.08 0.70 404 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.04 3.16 0.22 1.61 86.99 3.90 1.02 0.66 332 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 0.04 2.92 0.23 1.71 86.45 4.10 0.97 0.79 344 57-05 (1764-43-6x1975-90-14) avg 0.03 2.89 0.21 1.17 70.30 18.55 2.84 0.61 (n = 12) Vest-57 avg (n = 10) 0.04 4.17 0.23 1.67 77.79 11.35 0.91 0.83 Vest-70 avg (n = 12) 0.06 4.76 0.21 1.60 65.52 23.50 0.95 0.74 Research TOT ID Pedigree C20:1 C20:2 C22:0 C22:1 C24:0 C24:1 SATS GP #1- Vest-57x(1764-43-6x1975- 1.67 0.10 0.34 0.05 0.21 0.20 4.72 396 90-14) GP #1-24 Vest-57x(1764-43-6x1975- 1.60 0.07 0.33 0.04 0.17 0.30 5.08 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.55 0.07 0.32 0.04 0.19 0.22 5.15 181 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.72 0.07 0.40 0.04 0.27 0.27 5.16 444 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.68 0.08 0.34 0.03 0.18 0.25 5.20 449 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.64 0.10 0.38 0.06 0.25 0.24 5.32 240 90-14) GP #1- Vest-57x(1764-43-6x1975- 1.67 0.09 0.38 0.03 0.22 0.23 5.39 150 90-14) GP # 4- Vest-70x(1764-43-6x1975- 1.76 0.10 0.35 0.06 0.26 0.23 5.21 20 90-14) GP # 4- Vest-70x(1764-43-6x1975- 1.58 0.11 0.34 0.05 0.16 0.25 5.74 17 90-14) GP # 5- (01OB054RxLSAt15.36)xVest- 1.76 0.10 0.44 0.05 0.24 0.25 5.76 434 57-05 GP # 5-* (01OB054RxLSAt15.36)xVest- 1.92 0.05 0.52 0.05 0.29 0.26 5.80 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.71 0.06 0.34 0.04 0.19 0.18 6.04 351 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.69 0.10 0.41 0.05 0.31 0.26 6.05 334 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.74 0.08 0.49 0.05 0.29 0.18 6.08 404 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.44 0.05 0.44 0.05 0.27 0.15 6.18 332 57-05 GP # 5- (01OB054RxLSAt15.36)xVest- 1.76 0.07 0.48 0.04 0.24 0.20 6.19 344 57-05 (1764-43-6x1975-90-14) avg 1.92 0.15 0.50 0.12 0.35 0.37 5.54 (n = 12) Vest-57 avg (n = 10) 1.57 0.06 0.63 0.06 0.42 0.28 7.75 Vest-70 avg (n = 12) 1.40 0.09 0.52 0.06 0.33 0.28 8.01

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A Brassica napus seed designated 1904 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11273.
 2. A plant grown from the seed of claim 1, wherein said seed comprises a modified allele at a fatty acid desaturase 3E fad3E locus.
 3. A Brassica napus seed designated 2558 and represented by American Type Culture Collection (ATCC) Accession No. PTA-11274.
 4. A plant grown from the seed of claim 3, wherein said seed comprises a modified allele at a fad3E locus.
 5. The seed of the plant of claim 2, wherein said seed comprises a modified allele at a fad3E locus.
 6. The seed of the plant of claim 4, wherein said seed comprises a modified allele at a fad3E locus. 